Use of ixolaris, a tissue factor inhibitor, for the treatment and prevention of cancer

ABSTRACT

The invention provides methods for treatment of tissue factor (TF) mediated or associated diseases or processes, such as cancer, by administering at least an active fragment of an Ixolaris polypeptide to a subject. The invention further includes identification of a subject in need of such treatment, and monitoring a subject for amelioration of at least one sign or symptom of the disease. The invention also features kits.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.:U.S. Provisional Application No. 61/161,223, filed Mar. 18, 2009 and61/225,961, filed Jul. 16, 2009. The entire contents of theaforementioned provisional applications are hereby incorporated byreference.

GOVERNMENT SUPPORT

The following invention was supported at least in part by grant from theNational Institute of Health. Accordingly, the government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Malignant gliomas are very aggressive cancers, displaying high rates ofmortality (within months) and resistance to therapeutic interventions.Levels of tissue factor (TF) expression have been shown to correlatewith the histological grade of malignancy and vascularity in a number ofcancer types, including glioblastoma. Further, TF is overexpressedaround the typical necrotic foci found in glioblastoma [20]. Theseregions are highly hypoxic and seem to play a key role in glioblastomaaggressiveness, presenting an increased production of VEGF, IL-8 andmetalloproteases [21,22].

Ixolaris, a tick salivary 140 amino acid protein containing 10 cysteinesand 2 Kunitz-like domains, binds to FXa or FX as a scaffold forinhibition of the TF/FVIIa complex, in which the FVIIa catalytic site isinactivated. In contrast to TFPI, however, Ixolaris does not bind to theactive site cleft of FXa. Instead, complex formation is mediated by theFXa heparin-binding exosite [26]. In addition, Ixolaris interacts withzymogen FX through a precursor state of the heparin-binding exosite[27]. Because Ixolaris displays potent and long-lasting antithromboticactivity this molecule might interfere with glioblastoma progression.

Inhibition or targeting of TF may therefore provide an anti-tumorstrategy that could affect the survival of TF overexpressing tumor cellsby inhibiting TF mediated cellular signaling or other activities.Further, this approach may prevent tumor growth indirectly via anantiangiogenic mechanism by inhibiting the growth or function of TFexpressing intra-tumoral endothelial cells.

The present invention is directed to novel therapies for TF mediated orassociated diseases or processes, and in particular cancer and vasculardiseases.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on work by the presentinventors who found that the tick anticoagulant Ixolaris blocked tissuefactor (TF) dependent procoagulant activity of human glioblastoma andmelanoma cell lines and attenuated multimolecular coagulation complexassembly. The present inventors have found that Ixolaris inhibits invivo tumorigenic potential of human glioblastoma and melanoma cells innude mice, without observable bleeding, and was associated with reducedtumor vascularization and VEGF expression. Accordingly, the presentinventors describe Ixolaris as a promising agent for anti-tumor therapy.

In a first aspect, the present invention features a method of inhibitinggrowth of a cell expressing tissue factor (TF), comprising contactingthe cell with an effective amount of a tissue factor pathway inhibitor(TFPI) compound, such that the growth of the cell is inhibited.

In another aspect, the present invention features a method of treatingor preventing a TF mediated or associated disease or process in asubject, comprising administering to the subject a TFPI compound in anamount effective to treat or prevent the TF mediated or associateddisease or process.

In another further aspect, the present invention features a method oftreating or preventing the growth or metastasis of tumor cells in asubject, comprising administering to the subject a TFPI compound in anamount effective to treat or prevent the growth or metastasis of thetumor cells.

In one embodiment, the cells are selected from tumor cells, endothelialcells, vascular smooth muscle cells, inflammatory cells, or acombination thereof.

In another embodiment of the above aspects, the TFPI compound comprisesa tick saliva protein.

In another further embodiments, the TFPI compound comprises at least anactive fragment of an Ixolaris polypeptide.

In still another embodiments, the disease is selected from the groupconsisting of: cancer and a vascular disease.

In another embodiment, the tumor comprises a solid tumor.

In still another embodiment, the tumor comprises a central nervoussystem tumor or a squamous cell tumor.

In another embodiment, the tumor is a glioblastoma.

In still another embodiment, the tumor is a melanoma.

In another embodiment of any one of the above aspects, the tumor is ametastatic tumor.

In still another embodiment, treating or preventing the growth of tumorcells comprises at least one selected from the group consisting of:decreasing the rate of tumor growth, stopping tumor growth, shrinkingthe tumor, lessening tumor burden, preventing metastasis, or reducing atleast one sign or symptom associated with the presence of a tumor.

In another embodiment, the one or more tumor markers are a sign orsymptom associated with the presence of a tumor.

In another aspect, the invention features a method of treating orpreventing a vascular disease in a subject comprising administering tothe subject a TFPI compound in an amount effective to treat or preventthe vascular disease.

In one embodiment, the TFPI compound comprises a tick saliva protein.

In another embodiment, the TFPI compound comprises at least an activefragment of an Ixolaris polypeptide.

In still another embodiment, the vascular disease is selected fromdiabetic retinopathy, age-related macular degeneration, and pulmonaryhypertension.

In another embodiment, administering a tick saliva protein inhibitsangiogenesis in the subject.

In another preferred embodiment of any one of the above aspects, theTFPI compound is administered in combination concurrently orsequentially with another agent.

In another further embodiment, the agent is selected a cytotoxic agent,an anti-neoplastic agent, an immunosuppressive, and a VEGF antagonist.

In another preferred embodiment of any one of the above aspects, themethod further comprises identifying a subject in need of treatment witha TFPI compound.

In still another preferred embodiment of any one of the above aspects,the method further comprises monitoring a subject for effects oftreatment with TFPI compound.

In another aspect, the invention features a method of treating orpreventing cancer in a subject comprising administering to the subject aTFPI compound in an amount effective to treat or prevent cancer.

In one embodiment, the TFPI compound comprises a tick saliva protein.

In another embodiment, the TFPI compound comprises at least an activefragment of an Ixolaris polypeptide.

In a further embodiment of the above aspects, the method furthercomprises monitoring the subject for amelioration of at least one signor symptom of cancer.

In still another embodiment of the above aspects, the cancer comprises atumor with high expression or production of one or more proteinsselected from tissue factor (TF), Factor VIIa, Factor Xa, thrombin,vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), one ormore matrix metalloproteases, Factor VII, and Factor X, as compared to acontrol cell not derived from the tumor.

In another embodiment of the above aspects, the cancer comprises a tumorwherein the tumor expresses TF around the necrotic core.

In still another embodiment of the above aspects, the tumor is a highlyvascularized tumor.

In still another embodiment of the above aspects, the tumor comprises aglioblastoma.

In another embodiment of the above aspects, an active fragment of anIxolaris polypeptide comprises at least 40 contiguous amino acids ormore, 50 contiguous amino acids or more, 60 contiguous amino acids ormore, 70 contiguous amino acids or more, 80 contiguous amino acids ormore, 90 contiguous amino acids or more, 100 contiguous amino acids ormore, 110 contiguous amino acids or more, 120 contiguous amino acids ormore, 130 contiguous amino acids or more, 140 contiguous amino acids ormore, 150 contiguous amino acids or more, 160 contiguous amino acids ormore, or the full length sequence of the amino acid sequencecorresponding to SEQ ID NO: 2.

In another embodiment of the above aspects, an active Ixolarispolypeptide comprises at least 80% overall identity or more, 85% overallidentity or more, 90% overall identity or more, 95% overall identity ormore to a fragment of at least 50 contiguous amino acids of SEQ ID NO:2.

In another embodiment, amelioration of at least one sign or symptom ofcancer comprises at least one of a reduction in tumor volume or areduction of expression or production of at least one of tissue factor(TF), Factor VIIa, Factor Xa, thrombin, vascular endothelial growthfactor (VEGF), IL-8, one or more matrix metalloproteases, Factor VII, orFactor X as compared to prior to treatment with an Ixolaris polypeptide.

In another further embodiment, a reduction in tumor volume comprises areduction of 20% or more, 30% or more, 40% or more, 50% or more, 60% ormore, 70% or more, 80% or more, or 90% or more.

In still another embodiment of the above aspects, the active fragment ofan Ixolaris polypeptide comprises administration of a nucleic acidencoding the active fragment of an Ixolaris polypeptide operably linkedto control sequences for expression of the polypeptide.

In another embodiment of the above aspects, the method further comprisesadministration of a an agent for treatment of excess angiogenesis.

In still another embodiment of the above aspects, the method furthercomprises obtaining an Ixolaris polypeptide.

In another embodiment of the above aspects, the active fragment of theIxolaris polypeptide is administered at a daily dose of about 1 μg/kg toabout 1000 μg/kg, about 10 μg/kg to about 500 μg/kg, about 10 μg/kg toabout 750 μg/kg, about 25 μg/kg to about 1000 μg/kg, about 50 μg/kg toabout 1000 μg/kg, about 50 μg/kg to about 500 μg/kg, about 25 μg/kg toabout 500 μg/kg, or about 25 μg/kg to about 250 μg/kg.

In another further embodiment of the above aspects, the active fragmentof the Ixolaris polypeptide is administered one time or more, two timesor more, three times or more, four times or more, five times or more,six times or more, seven times or more, eight times or more, ten timesor more, fifteen times or more, twenty times or more, or twenty fivetimes or more.

In still another embodiment of the above aspects, the Ixolarispolypeptide is an isolated polypeptide.

The invention also features in preferred embodiments, a pharmaceuticalcomposition for practicing any of the methods of the aspects describedherein.

In another aspect, the invention features a pharmaceutical compositioncomprising an isolated Ixolaris polypeptide in a pharmaceuticalexcipient.

In another further embodiment, the invention features an antibody thatrecognizes one or more epitope of a TFPI compound.

In one embodiment, the TFPI compound comprises a tick saliva protein.

In another embodiment, the TFPI compound comprises at least an activefragment of an Ixolaris polypeptide.

In another aspect, the invention provides a kit for practicing any ofthe methods of the aspects described herein, and instructions for use.

In one embodiment, the kit comprises a TFPI compound.

In another embodiment, the TFPI compound comprises a tick salivaprotein.

In another further embodiment, the TFPI compound comprises at least anactive fragment of an Ixolaris polypeptide.

DESCRIPTION OF THE DRAWINGS

FIG. 1(A-C) are graphs that show functional TF expressed by U87-MG cellsis inhibited by Ixolaris. (A) Expression of TF on U87-MG (left) andMDA-MB-231 (right) cells was evaluated by flow-cytometric analysis.Dashed line represents staining with monoclonal anti-human TF antibody,followed by FITC-conjugated secondary antibody. Grey region representscontrol in the absence of primary antibody. (B) Assembly of extrinsictenase complex on U87-MG cells. Kinetics for the activation of FX (100nM) in the presence of FVIIa (1 nM) and U87-MG cells (5×10⁵ mL⁻¹) ().Controls were performed in the presence of cells (5×10⁵ mL⁻¹) andabsence of FVIIa (▪) or in the absence of cells and presence of FVIIa (1nM) (▴). (C) Inhibitory effect of Ixolaris. FX (100 nM) was incubatedfor 5 min with the indicated concentrations of Ixolaris prior toactivation by FVIIa (1 nM) and U87-MGcells (5×10⁵ mL⁻¹). The conditionsfor the assays and for quantification of Xa are described in theMaterials and methods section. Each point represents mean±SD of threedeterminations.

FIG. 2(A-F) are four graphs that show ixolaris inhibits PS-dependentprocoagulant complexes assembled on U87-MG cells. Assembly ofPS-dependent procoagulant complexes on U87-MG cells. (A) PS exposure onU87-MG was evaluated by flow-cytometric analysis of annexin V binding tocells. Dashed line represents staining with FITC-labeled annexin V. Greyregion represents control performed in the absence of annexin V. (B)Assembly of intrinsic tenase complex on U87-MG cells. Kinetics for theactivation of FX (100 nM) in the presence of FIXa (0.2 nM), FVIIIa (4 IUmL⁻¹) and () U87-MG cells (5×10⁵ mL⁻¹). Controls were performed in theabsence of cells (s). Assay conditions and quantification of Xa aredescribed in the Materials and methods section. Each point representsmean±SD of three determinations. (C) Prothrombinase complex assembly onU87-MG cells. (A) Kinetics for the activation of prothrombin (0.5 lM) inthe presence of FXa (10 pM), FVa (1 nM) and U87-MG cells (5·105 cellsmL⁻¹ (▪). Controls were performed in the absence of cells (□). Assayconditions and quantification of thrombin are described in the Materialsand methods section. Each point represents mean±SD of threedeterminations. (D) Inhibitory effect of annexin V on FX () orprothrombin activation (▪). U87-MG cells (5×10⁵ mL⁻¹) were incubatedwith the indicated concentrations of annexin V prior to addition ofeither FXa (10 pM)/prothrombin (0.5 IM) or FIXa (0.2 nM)/FX (100 nM),followed by addition of FVa (1 nM) or FVIIIa (4 IU mL⁻¹), respectively.Zymogen activation rates in the absence of annexin V were taken as 100%.Assay conditions are described in the Materials and methods section.Each point represents mean±SD of three determinations. (E) Activation ofFX (100 nM) by FIXa (0.2 nM), FVIIIa (4 U mL⁻¹) and U87-MG cells (5×10⁵mL⁻¹) was assayed at the indicated concentrations of Ixolaris. (F)Prothrombin (0.5 μM) activation by FXa (10 μM) in the presence of FVa (1nM) and U87-MG cells (5×10⁵ mL⁻¹) was assayed at the indicatedconcentrations of Ixolaris. Assay conditions are described in theMaterials and methods section. Each point represents mean±SD of threedeterminations.

FIGS. 3(A and B) shows procoagulant activity of U87-MG cells is reversedby Ixolaris. (A) U87-MG cells (in PBS) at the indicated concentrationswere incubated with human plasma followed by recalcification with 12.5mM CaCl₂. Each point represents mean±SD of three assays. (B) Humanplasma was incubated for 5 min with the indicated concentrations ofDEGR-FVIIa (FVIIai, grey bars) or Ixolaris (black bars) prior toaddition of U87-MG cells (5×10⁵ cells) followed by recalcification with12.5 mM CaCl₂. Each point represents mean±SD of three assays.

FIGS. 4(A and B) shows Ixolaris inhibits in vivo primary tumor growth ina xenograft model. U87-MG cells were injected subcutaneously (s.c.) intonude mice. Treatment with Ixolaris was initiated 3 days after tumor cellinoculation; control animals were treated with an equivalent volume ofPBS. (A) Tumor size was measured at the indicated days. Each pointrepresents mean±SD. (B) After 20 days of tumor cell inoculation, animalswere sacrificed and tumors were removed and weighed.

FIG. 5(A-C) shows treatment with Ixolaris decreases tumor angiogenesis.(A) RNA was extracted from tumors obtained from experiments depicted inFIG. 4(B) and further analyzed for VEGF expression using RT-PCR, asdescribed in the Materials and methods section. (B) Bar graph showsdecreased VEGF expression in Ixolaris-treated animals (n=5; 10.4±3.6)compared with PBS-treated controls (n=5; 22.7±2.6). VEGF staining(asterisk) and quantification was performed as described in theMaterials and methods section. (C) Bar graph shows that there are fewerblood vessels in Ixolaris-treated animals (n=5; 8.9±3.7) than inPBS-treated controls (n=5; 23.9±4.2). Vessel density was evaluated inCD105-stained tumor sections (arrows) as described in the Materials andmethods section. Values of P<0.05 were considered to be statisticallysignificant. All values are given as mean±SD.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press;Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by EdwardHarlow, David Lane, Ed Harlow (1999, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow(Editor), David Lane (Editor) (1988, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-3,4-2), 1855. Handbook of Drug Screening, edited byRamakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y.,Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes,Reagents, and Other Reference Tools for Use at the Bench, Edited JaneRoskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN0-87969-630-3. Each of these general texts is herein incorporated byreference.

As used herein, the following terms have the meanings ascribed to thembelow, unless specified otherwise.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value.

As used herein, the term “cancer” is used to mean a condition in which acell in a patient's body undergoes abnormal, uncontrolled proliferation.Thus, “cancer” is a cell-proliferative disorder. “Cancer” as used hereinis understood as any of a class of diseases in which a group of cellsdisplay uncontrolled growth (division beyond the normal limits),invasion (intrusion on and destruction of adjacent tissues), andsometimes metastasis (spread to other locations in the body via lymph orblood). Cancers can be divided into two large groups of solid tumors andnon-solid tumors (e.g., blood tumors such as leukemias). Cancer canoccur in nearly any tissue of the body including, but not limited toadrenocortical carcinoma, anal cancer, bladder cancer, brain stemglioma, brain tumors, breast cancer, cerebellar astrocytoma/malignantglioma, cervical cancer, chronic myeloproliferative disorders, coloncancer, endometrial cancer, ependymoma, esophageal cancer, Ewing familyof tumors, extracranial germ cell tumors, extragonadal germ cell tumors,extrahepatic bile duct cancer, gallbladder cancer, gastric cancer,gastrointestinal carcinoid tumors, gestational trophoblastic tumors,hypopharyngeal cancer, islet cell carcinoma (Endocrine Pancreas), isletcell tumors (endocrine pancreas), laryngeal cancer, leukemia, acutelymphoblastic; leukemia, acute myeloid; leukemia, chronic lymphocytic;leukemia, chronic Myelogenous, lip and oral cavity cancer, liver cancer,lung cancer, non-small cell; lung cancer, small cell, lymphoma,Hodgkin's, lymphoma, Non-Hodgkin's, lymphoma, AIDS-Related, lymphoma,lymphoma, primary CNS, melanoma, intraocular (Eye), medulloblastoma,Merkel cell carcinoma, mesothelioma, mycosis fungoides and the SézarySyndrome, myelodysplastic and myeloproliferative diseases,myelodysplastic Syndromes, myeloid leukemia/other myeloid cancers,nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer,osteosarcoma/malignant fibrous histiocytoma of bone, ovarian epithelialcancer, ovarian germ cell tumors, pancreatic cancer, paranasal sinus andnasal cavity cancer, parathyroid cancer, penile cancer,pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer,renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary glandcancer, sarcoma, soft Tissue, sarcoma, Kaposi, skin cancer, smallIntestine cancer, squamous neck cancer with occult primary,supratentorial primitive neuroectodermal tumors and pineoblastoma,testicular cancer, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter, urethralcancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamicglioma, and vulvar cancer.

As used herein “amelioration” or “treatment” is understood as meaning tolessen or decrease at least one sign, symptom, indication, or effect ofa specific disease or condition. For example, amelioration or treatmentof cancer can include preventing of the formation of tumors, shrinkingone or more tumors, limiting or preventing the formation of metastases.As used herein, “prevention” is understood as to delay, limit, reducethe rate or degree of onset, or inhibit the development of at least onesign or symptom of a disease or condition. Prevention, amelioration, andtreatment can require administration of one or more doses of at least anactive fragment of an ixolaris polypeptide.

As used herein, “contacting a cell” is meant to refer to providing anagent to a cell, in culture or in an animal, such that the agent caninteract with the surface of the cell, potentially be taken up by thecell, and have an effect on the cell. The agent can be delivered to thecell directly (e.g., by addition of the agent to culture medium or byinjection into the cell or tissue of interest), or by delivery to theorganism by an enteral or parenteral route of administration fordelivery to the cell by circulation, lymphatic, or other means.

As used herein, “detecting”, “detection” and the like are understoodthat an assay performed for identification of a specific analyte in asample or a change in a subject of at least one sign or symptom of adisease, expression of a protein or gene, including a reporterconstruct. The amount of analyte detected in the sample or change ofbehavior in a subject can be none or below the level of detection of theassay or method.

As used herein, the terms “effective” and “effectiveness” includes bothpharmacological effectiveness and physiological safety. Pharmacologicaleffectiveness refers to the ability of the treatment to result in adesired biological effect in the patient. Physiological safety refers tothe level of toxicity, or other adverse physiological effects at thecellular, organ and/or organism level (often referred to asside-effects) resulting from administration of the treatment. On theother hand, the term “ineffective” indicates that a treatment does notprovide sufficient pharmacological effect to be therapeutically useful,even in the absence of deleterious effects, at least in the unstratifiedpopulation. (Such a treatment may be ineffective in a subgroup that canbe identified by the expression profile or profiles.) “Less effective”means that the treatment results in a therapeutically significant lowerlevel of pharmacological effectiveness and/or a therapeutically greaterlevel of adverse physiological effects, e.g., greater liver toxicity.

Thus, in connection with the administration of a drug, a drug which is“effective against” a disease or condition indicates that administrationin a clinically appropriate manner results in a beneficial effect for atleast a statistically significant fraction of patients, such as aimprovement of symptoms, a cure, a reduction in disease signs orsymptoms, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating the particular type of disease or condition.

As used herein, “isolated” or “purified” when used in reference to apolypeptide means that a naturally polypeptide or protein has beenremoved from its normal physiological environment (e.g., proteinisolated from plasma or tissue) or is synthesized in a non-naturalenvironment (e.g., artificially synthesized in a heterologous system).Thus, an “isolated” or “purified” polypeptide can be in a cell-freesolution or placed in a different cellular environment (e.g., expressedin a heterologous cell type). The term “purified” does not imply thatthe polypeptide is the only polypeptide present, but that it isessentially free (about 90-95%, up to 99-100% pure) of cellular ororganismal material naturally associated with it, and thus isdistinguished from naturally occurring polypeptide. “Isolated” when usedin reference to a cell means the cell is in culture (i.e., not in ananimal), either cell culture or organ culture, of a primary cell or cellline. Cells can be isolated from a normal animal, a transgenic animal,an animal having spontaneously occurring genetic changes, and/or ananimal having a genetic and/or induced disease or condition. Isolatedcells can be further modified to include reporter constructs or betreated with various stimuli to modulate expression of a gene ofinterest.

As used herein, “ixolaris” is understood as the amino acid andpolypeptide sequence provided at GenBank Accession No. (GenBank:AF286029.1) and described in US Patent Publication 20040018516(incorporated herein by reference). At least an active fragment of anixolaris polypeptide is understood as a polypeptide that has at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95% of the inhibitory activityof a wild-type, full length ixolaris polypeptide or a mature processed140 amino acid ixolaris polypeptide in a TF-mediated coagulant activityassay such as that provided herein, or any of the other assays providedherein to test ixolaris activity such as Factor X generation assay, thethrombin generation assay, the inhibition of formation of coagulationcomplexes by annexin V, or modification of coagulation time. An activefragment of an ixolaris polypeptide includes at least 40 contiguousamino acids, 50 contiguous amino acids, 60 contiguous amino acids, 70contiguous amino acids, 80 contiguous amino acids, 90 contiguous aminoacids, 100 contiguous amino acids, 110 contiguous amino acids, 120contiguous amino acids, 130 contiguous amino acids, 140 contiguous aminoacids, 150 contiguous amino acids, 160 contiguous amino acids of theamino acid sequence provided by GenBank: AF286029.1. An active ixolarispolypeptide has at least 80%, at least 85%, at least 90%, at least 95%overall identity to a fragment of at least 50 contiguous amino acids ofGenBank: AF286029.1.

As used herein, “kits” are understood to contain at least thenon-standard laboratory reagents for use in the methods of theinvention, such as a ixolaris polypeptide or amino acid coding sequence.The kit can further include any other components required to practicethe method of the invention, as dry powders, concentrated solutions, orready to use solutions. In some embodiments, the kit comprises one ormore containers that contain reagents for use in the methods of theinvention; such containers can be boxes, ampules, bottles, vials, tubes,bags, pouches, blister-packs, or other suitable container forms known inthe art. Such containers can be made of plastic, glass, laminated paper,metal foil, or other materials suitable for holding reagents.

As used herein, the term “obtaining” is understood to refer tomanufacturing, purchasing, or otherwise coming into possession of.

“Peptide”, “polypeptide”, “protein”, and the like are understood as twoor more naturally occurring or synthetic amino acids joined by an amidelinkage. Optionally the peptide further includes one or moremodifications such as modified peptide bonds, i.e., peptide isosteres,and may contain amino acids other than the 20 gene-encoded amino acids.The polypeptides may be modified by either natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in apolypeptide, including the peptide backbone, the amino acid side-chainsand the amino or carboxyl termini. It will be appreciated that the sametype of modification may be present in the same or varying degrees atseveral sites in a given polypeptide. Also, a given polypeptide maycontain many types of modifications. Polypeptides may be branched, forexample, as a result of ubiquitination, and they may be cyclic, with orwithout branching. Cyclic, branched, and branched cyclic polypeptidesmay result from posttranslation natural processes or may be made bysynthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formulation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, Proteins, Structure and Molecular Properties, 2nded., T. E. Creighton, W.H. Freeman and Company, New York (1993);Posttranslational Covalent Modification of Proteins, B. C. Johnson, ed.,Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.Enzymol 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci.663:48-62 (1992)).

As used herein, the phrase “pharmaceutically acceptable carrier” is artrecognized and includes a pharmaceutically acceptable material,composition or vehicle, suitable for administering compounds of thepresent invention to mammals. The carriers include liquid or solidfiller, diluent, excipient, solvent or encapsulating material, involvedin carrying or transporting the subject agent from one organ, or portionof the body, to another organ, or portion of the body. Each carrier mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the patient. Someexamples of materials which can serve as pharmaceutically acceptablecarriers include: sugars, such as lactose, glucose and sucrose;starches, such as corn starch and potato starch; cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients,such as cocoa butter and suppository waxes; oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical formulations.

As used herein, the term “subject” is meant to refer to livingorganisms. In certain embodiments, the living organism is an animal. Incertain preferred embodiments, the subject is a mammal. In certainembodiments, the subject is a domesticated mammal. Examples of subjectsinclude humans, non-human primates, dogs, cats, mice, rats, cows,horses, goats, and sheep. A human subject may also be referred to as apatient.

A subject “suffering from or suspected of suffering from” a specificdisease, condition, or syndrome has at least one risk factor or presentswith at least one sign or symptom of the disease, condition, or syndromesuch that a competent individual would diagnose or suspect that thesubject was suffering from the disease, condition, or syndrome. Methodsfor identification of subjects suffering from or suspected of sufferingfrom cancer is within the ability of those in the art. Methods ofidentifying specific genetic or lifestyle predispositions to cancer iswell within the ability of those of skill in the art. Subjects sufferingfrom, and suspected of suffering from, a specific disease, condition, orsyndrome are not necessarily two distinct groups.

As used herein, the term “therapeutically effective amount,” is meant torefer to an amount of an agent which is effective, upon single ormultiple dose administration to the cell or subject, decreasing at leastone sign or symptom of the disease or disorder, or prolonging thesurvivability of the patient with such a disease or disorder beyond thatexpected in the absence of such treatment.

As used herein, the term “tissue factor (TF) protein” is meant to referto a polypeptide having an amino acid sequence corresponding to anaturally occurring tissue factor, and particularly a mammalian tissuefactor or a recombinant tissue factor. Naturally occurring TF includeshuman species as well as other animal species such as rabbit, rat,porcine, non human primate, equine, murine, and ovine tissue factor(see, for example, Hartzell et al., (1989) Mol. Cell. Biol.,9:2567-2573; Andrews et al., (1991) Gene, 98:265-269; and Takayenik etal., (1991) Biochem. Biophys. Res. Comm., 181:1145-1150). In certainembodiments, the amino acid sequence of human tissue factor correspondsto NCBI Accession No. NP_(—)001984.1 and is represented by SEQ ID NO:1-. The amino acid sequences of the other mammalian tissue factorproteins are generally known or obtainable through conventionaltechniques.

As used herein, “tissue factor (TF) mediated or associated disease orprocess” according to the present invention is any event which ismediated by the presence of TF. A “TF related disease” is meant to referto a disease or disorder which may be impacted through the inhibition ofTF, particularly the inhibition of tumor growth on tissue factorexpressing cells, but also includes other tissue factor mediateddiseases such as chronic thromboembolic diseases or disorders associatedwith fibrin formation including vascular disorders such as deep venousthrombosis, arterial thrombosis, stroke, tumor metastasis, thrombolysis,arteriosclerosis and restenosis following angioplasty, acute and chronicindications such as inflammation, septic shock, septicemia, hypotension,adult respiratory distress syndrome (ARDS), disseminated intravascularcoagulopathy (DIC) and other diseases.

As used herein, the term “subject” includes any human or non-humananimal. The term “non-human animal” includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dog, cow,chickens, amphibians, reptiles, etc.

As used herein, the term “vascular disease” is meant to refer to anydisease or disorder that affects the circulatory system.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Tissue Factor (TF)

The coagulation of blood involves a cascading series of reactionsleading to the formation of fibrin. The coagulation cascade consists oftwo overlapping pathways, both of which are required for hemostasis. Theintrinsic pathway comprises protein factors present in circulatingblood, while the extrinsic pathway requires tissue factor (TF), which isexpressed on the cell surface of a variety of tissues in response tovascular injury. When exposed to blood, TF sets in motion a cascade ofactivation steps that result in the formation of an insoluble fibrinclot.

TF has been investigated as a target for anticoagulant therapy. TF is asingle chain, 263 amino acid membrane glycoprotein that functions as areceptor for factor VII and VIIa and thereby initiates the extrinsicpathway of the coagulation cascade in response to vascular injury. TF isa transmembrane cell surface receptor which serves as the receptor aswell as the cofactor for factor VIIa, forming a proteolytically activeTF:VIIa complex on cell surfaces.

In certain embodiments, the amino acid sequence of human tissue factor(precursor) corresponds to NCBI Accession No. NP_(—)001984.1 and isrepresented by SEQ ID NO: 1, shown below:

SEQ ID NO: 1 metpawprvp rpetavartl llgwvfaqva gasgttntvaaynltwkstn fktilewepk pvnqvytvqi stksgdwkskcfyttdtecd ltdeivkdvk qtylarvfsy pagnvestgsageplyensp eftpyletnl gqptiqsfeq vgtkvnvtvedertlvrrnn tflslrdvfg kdliytlyyw kssssgkktaktntneflid vdkgenycfs vqavipsrtv nrkstdspvecmgqekgefr eifyiigavv fvviilviil aislhkcrka gvgqswkens plnvs

Vessel wall injury leads to exposure of membrane-bound tissue factor(TF), which is a crucial step in the initiation of blood coagulation[1]. TF functions as a cofactor for blood coagulation factor VIIa(FVIIa), and the resultant binary FVIIa/TF complex then generates FIXaand FXa. Generation of FIXa by the FVIIa/TF complex results in formationof the tenase complex, following binding to the non-enzymatic co-factor,activated FVIIIa. The tenase complex, along with FVIIa/TF, converts FXto FXa, which assembles with FVa into the prothrombinase complex that isdirectly responsible for the formation of thrombin [2,3].

Constitutive tissue distribution of TF is highly heterogeneous [4] andits induced and/or deregulated expression has been related to a numberof pathological processes [5,6].

Tissue factor is overexpressed on a variety of malignant tumors andisolated human tumor cell lines, suggesting a role in tumor growth andsurvival. Abnormal elevated TF expression has been well documented inseveral tumor types, and seems to be directly correlated withthromboembolic complications in cancer patients [7,8]. TF is notproduced by healthy endothelial cells lining normal blood vessels but isexpressed on these cells in tumor vessels. It appears to play a role inboth vasculogenesis, the formation of new blood vessels in thedeveloping animal and in angiogenesis, the sprouting of new capillariesfrom existing arteries, in normal and malignant adult tissues.

Aberrant expression of TF on endothelial and tumor cells in a variety ofbreast, colorectal, lung and pancreatic cancers has been linked to anincrease in tumor microvessel density and upregulated VEGF expression.Studies employing cultured cells as well as patients' specimens havedemonstrated strong correlation between TF expression and aggressivetumor behavior [9-12]. In particular, TF expression correlates with anunbalanced production of anti- and/or proangiogenic factors, such asvascular endothelial growth factor (VEGF), thus favoring increased tumorvascularity [13-16]. Tumor cells over expressing TF are also thought tobe responsible for the thrombotic complications associated with cancer.

Pro-tumoral effects of TF and blood clotting enzymes (FVIIa, FXa andthrombin) are intimately related to a group of G protein-coupledreceptors named Protease Activated Receptors (PARs). In fact, activationof PARs in cancer cells elicits a vast number of cellular responses,which include migration, invasion, proliferation, metastasis, inhibitionof apoptosis, and production of several proaggressive factors such asVEGF, interleukin-8 (IL-8), metalloproteases and others [17,18].

Ixolaris Polypeptides and Nucleic Acids

Saliva of the hard tick, Ixodes scapularis, has a repertoire ofcompounds that counteracts host defenses. The present inventors havepreviously cloned and expressed the tick tissue factor pathwayinhibitor, Ixolaris. Recombinant Ixolaris is a highly specific inhibitorof the extrinsic pathway that blocks generation of Xa by TF/VIIa with anapparent Ki in the pM range (US Patent Application 20040018516,incorporated by reference in its entirety herein).

Ixolaris was cloned from a salivary gland cDNA library of the tickIxodes scapularis was randomly cloned and sequenced, identifying a cDNAwith high similarity to rabbit tissue factor pathway inhibitor. Thefull-length nucleotide and deduced amino acid sequences of IxodesTFPI-like protein are provided in SEQ ID NO: 470 and SEQ ID NO: 471respectively in US Patent publication 20040018516. The translatedprotein has a short hydrophobic sequence of 25 amino acids typical ofsignal peptide and an alanine at the N-terminus, according to Signal Psoftware for prediction of N-terminal of proteins (Nielsen, H. et al.1997 Protein Eng 10:1-6). The mature protein contains 140 amino acids(15.7 kDa) (SEQ ID NO: 138 of US Patent publication 20040018516),including 10 cysteines and a pI of 4.56. Ixolaris is similar to othermembers of the Kunitz family of proteins including human TFPI precursor(e value=4⁻¹⁴, P10646); lacunin from Manduca sexta (1⁻¹², AAF04457.1);hepatocyte growth factor pathway inhibitor (8⁻¹², AAF02490.1);inter-.alpha.-trypsin inhibitor (bikunin) (7⁻¹¹, P04365);amyloid-precursor-like protein (1⁻¹¹, CAA54906.1); and basic pancreatictrypsin inhibitor (aprotinin, 1⁻⁰⁵, 1510193A).

Ixolaris potently inhibits factor VIIa/TF-induced Factor X activationwith an IC₅₀ in the pM range. Ixolaris is functionally and structurallydistinct from its endogenous counterpart, TFPI (53-55): although the sixcysteines that characterize the first Kunitz domain (55) of TFPI areconserved in Ixolaris, only four of six cysteines present in the secondKunitz domain of human TFPI are present. Also, whereas the sixth and thefirst cysteines that, respectively, terminate and initiate the first andsecond Kunitz domains in human TFPI are separated by 20 amino acids,only 7 amino acids separate the corresponding cysteines in Ixolaris.Additionally, the Kunitz-type domain 2 in Ixolaris is unusual bycontaining 4 additional amino acids between the fourth and fifthcysteine residues, making this loop longer than most Kunitz-type familymembers. Also, the presumed P1 reactive-site residue of the first domainin Ixolaris is Glu, whereas Lys occupies this position in TFPI (53).Ixolaris has a short and basic carboxy terminus but, unlike TFPI, it hasonly 14 amino acids where the positively charged amino acids are notorganized as a cluster. In human TFPI, this basic carboxy terminus hasbeen consistently shown to increase its anticoagulant activity (56, 57)and to shorten its half-life (57). The Ixolaris cDNA also encodes threeputative N-linked glycosylation sites, at Asn65, Asn98, and Asn136.Consistent with a calculated mass of 15.7 kDa for the carbohydrate-freeprotein, we could detect a band of about 15.5 kDa in the gels loadedwith recombinant Ixolaris; however, an intense smear was observed inPAGE of Ixolaris at a molecular weight range of about 24 kDa.Accordingly, it is likely that these Asn residues are glycosylated, andthis is the most abundant form (>95%) of the secreted recombinantmolecule.

In certain embodiments, the amino acid sequence of Ixodes scapularisTFPI is shown below and is represented by SEQ ID NO: 2, shown below,corresponding to Genbank Accession AF286029:

SEQ ID NO: 2 MRAVSCFLYYGVAWIALGSWGASSSAERVSEMDIYEFESWVSCLDPEQVTCESQEGTHASYNRKTGQCEEQKGTECGGGENHFETLLKCNES CNDAPKPPCSLEVDYGVGRANIPRWYYDTNNATCEMFTYGGITGNKNNFESEEECKET CKGFSLLKKV NVTIN

The nucleic acid sequence that encodes SEQ ID NO: 2 is shown below, andcorresponds to SEQ ID NO: 3.

SEQ ID NO: 3 atgcgcgctg tttcctgctt cctatattat ggagttgcttggattgcact tggaagttgg ggtgcgtcaa gttcagcagaacgtgttagc gaaatggaca tctatgagtt cgaatcctgggtatcttgtc ttgatcccga acaagtaacg tgtgaaagccaagagggaac gcacgcttca tacaaccgaa aaacgggacagtgtgaagag caaaagggaa cagagtgtgg aggaggcgagaatcactttg aaactttgtt gaagtgcaac gaatcttgcaacgatgctcc gaagccacct gctcgctgg aagtagattatggtgttgga agagctaaca taccacgatg gtattatgacaccaacaatg caacttgcga aatgttcacc tatgggggaataactggcaa taaaaacaat tttgaatccg aggaagagtgtaaggaaact tgcaagggtt tttctctgtt aaagaaagta aatgtcacta ttaactga

In addition to nucleotide sequences encoding the full-length Ixolarisprotein, the present invention may also include nucleotide sequencesencoding truncations of the Ixolaris protein that have the desiredeffects, for example prevent a TF mediated or associated disease orprocess, prevent the growth or metastasis of tumor cells. The nucleotidesequences encoding truncations of the Ixolaris protein preferablyexhibit binding affinity for Factor Xa, X, or VIIa, with the resultingbiological effect of Factor Xa, X, or VIIa binding, anticoagulantactivity, generation of antibodies that specifically bind Ixolaris, oridentification of compounds that can be used to modulate coagulationfunction.

Functional equivalents of Ixolaris include those naturally occurring andengineered, as judged by any of a number of criteria, including, but notlimited to, the binding affinity for Factor Xa, X, or VIIa, theresulting biological effect of factor Xa, X, or VIIa binding,anticoagulant activity, generation of antibodies that specifically bindIxolaris, and identification of compounds that can be used to in themethods of the present invention.

Truncations of Ixolaris preferably comprise an active fragment of anIxolaris polypeptide.

In addition to the Ixolaris nucleotide sequences described above, fullor partial length Ixolaris cDNA present in the same species and/orhomologs of the Ixolaris gene present in other species can be identifiedand readily isolated, without undue experimentation, by molecularbiological techniques well known in the art. For example, expressionlibraries of cDNAs synthesized from salivary gland mRNA derived from theorganism of interest can be screened using labeled Factor Xa, X, or VIIaderived from that species, e.g., a Factor Xa, X, or VIIa fusion protein.Alternatively, such cDNA libraries, or genomic DNA libraries derivedfrom the organism of interest can be screened by hybridization using thenucleotides described herein as hybridization or amplification probes.Furthermore, genes at other genetic loci within the genome that encodeproteins which have extensive homology to one or more domains of theIxolaris gene product can also be identified via similar techniques. Inthe case of cDNA libraries, such screening techniques can identifyclones derived from alternatively spliced transcripts in the same ordifferent species.

Screening can be by filter hybridization, using duplicate filters. Thelabeled probe can contain at least 15-30 base pairs of the Ixolarisnucleotide sequence, SEQ ID NO: 3. The hybridization washing conditionsused should be of a lower stringency when the cDNA library is derivedfrom an organism different from the type of organism from which thelabeled sequence was derived. With respect to the cloning of a humanIxolaris homolog, using tick Ixolaris probes, for example, hybridizationcan, for example, be performed at 65 C overnight in Church's buffer (7%SDS, 250 mM NaHPO4, 2 μM EDTA, 1% BSA). Washes can be done with 2 timesSSC, 0.1% SDS at 65 C. and then at 0.1 times SSC, 0.1% SDS at 65 C.

Low stringency conditions are well known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding these and other hybridization conditions see, for example,Sambrook et al. 1989 Molecular Cloning, A Laboratory Manual, ColdSprings Harbor Press, N.Y.; Ausubel et al. 1989 Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y.

Alternatively, the labeled Ixolaris nucleotide probe may be used toscreen a genomic library derived from the organism of interest, again,using appropriately stringent conditions.

Further, a full or partial length Ixolaris cDNA present in the samespecies and/or homologs of the Ixolaris gene present in other speciesmay be isolated from nucleic acid of the organism of interest byperforming PCR using two degenerate oligonucleotide primer poolsdesigned on the basis of amino acid sequences within the Ixolaris geneproduct disclosed herein. The template for the reaction may be cDNAobtained by reverse transcription of mRNA prepared from, for example,cell lines or tissue, such as salivary gland, known or suspected toexpress an Ixolaris gene allele.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of an Ixolaris gene. The PCRfragment may then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment may be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment may be used to isolate genomicclones via the screening of a genomic library.

PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express the Ixolaris gene, such as, for example,salivary gland). A reverse transcription reaction may be performed onthe RNA using an oligonucleotide primer specific for the most 5′ end ofthe amplified fragment for the priming of first strand synthesis. Theresulting RNA/DNA hybrid may then be “tailed” with guanines using astandard terminal transferase reaction, the hybrid may be digested withRNAase H, and second strand synthesis may then be primed with a poly-Cprimer. Thus, cDNA sequences upstream of the amplified fragment mayeasily be isolated. For a review of cloning strategies which may beused, see e.g., Sambrook et al. 1989, supra.

In certain preferred embodiments, an active fragment of an Ixolarispolypeptide comprises at least 40 contiguous amino acids or more, 50contiguous amino acids or more, 60 contiguous amino acids or more, 70contiguous amino acids or more, 80 contiguous amino acids or more, 90contiguous amino acids or more, 100 contiguous amino acids or more, 110contiguous amino acids or more, 120 contiguous amino acids or more, 130contiguous amino acids or more, 140 contiguous amino acids or more, 150contiguous amino acids or more, 160 contiguous amino acids or more, orthe full length sequence of the amino acid sequence corresponding to SEQID NO: 2.

In other embodiments, an active Ixolaris polypeptide comprises at least80% overall identity or more, 85% overall identity or more, 90% overallidentity or more, 95% overall identity or more to a fragment of at least50 contiguous amino acids of SEQ ID NO: 2.

The invention encompasses nucleotide sequences that encode not onlyIxolaris but also its functional domains, besides truncations thereof,as well as substitutions, insertions, and deletions (including fusionproteins) thereof. These include, but are not limited to nucleotidesequences encoding a Kunitz domain of the Ixolaris protein. It isbelieved that a Kunitz domain may be responsible for the observedanticoagulant activity. Certain representative Kunitz domains includebetween amino acids 18 and 68 (first Kunitz domain) and its amino andcarboxy truncations.

Amino acid substitutions may be of a conserved or non-conserved nature.Conserved amino acid substitutions consist of replacing one or moreamino acids of the Ixolaris or Ixolaris-related sequence with aminoacids of similar charge, size, and/or hydrophobicity characteristics,such as, for example, a glutamic acid (E) to aspartic acid (D) aminoacid substitution. Non-conserved substitutions consist of replacing oneor more amino acids of the Ixolaris or Ixolaris-related sequence withamino acids possessing dissimilar charge, size, and/or hydrophobicitycharacteristics, such as, for example, a glutamic acid (E) to valine (V)substitution. For example, nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine; polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;positively charged (basic) amino acids include arginine, lysine, andhistidine; and negatively charged (acidic) amino acids include asparticacid and glutamic acid. One or more such substitutions may be introducedinto the Ixolaris or Ixolaris-related sequence, as long as suchsubstitutions result in variants which exhibit binding affinity forFactor Xa, X, or VIIa, the resulting biological effect of Factor Xa, X,or VIIa binding, anticoagulant activity, generation of antibodies thatspecifically bind Ixolaris, or identification of compounds that can beused to modulate coagulation function.

Amino acid insertions may consist of single amino acid residues orstretches of residues. The insertions may be made at the carboxy oramino terminal end of the Ixolaris or Ixolaris-related sequence, as wellas at a position internal to the sequence. Such insertions made ateither the carboxy or amino terminus of the sequence of interest may beof a broader size range. One or more such insertions may be introducedinto the Ixolaris or Ixolaris-related sequence, as long as suchinsertions result in variants which exhibit binding affinity for FactorXa, X, or VIIa, the resulting biological effect of Factor Xa, X, or VIIabinding, anticoagulant activity, generation of antibodies thatspecifically bind Ixolaris, or identification of compounds that can beused to modulate coagulation function.

Deletions of Ixolaris or Ixolaris-related sequences are also within thescope of the invention. Such deletions consist of the removal of one ormore amino acids from the Ixolaris or Ixolaris-related sequence. Suchdeletions may involve a single contiguous or greater than one discreteportion of the original sequences. One or more such deletions may beintroduced into the Ixolaris or Ixolaris-related sequence, as long assuch deletions result in variants which exhibit binding affinity forFactor Xa, X, or VIIa, the resulting biological effect of Factor Xa, X,or VIIa binding, anticoagulant activity, generation of antibodies thatspecifically bind Ixolaris, or identification of compounds that can beused to modulate coagulation function.

The preferred amino acid sequences of the present invention are isolatedfrom their natural source by any of the methods known in the art. Thesemethods include preparing a soluble extract and enriching the extractusing chromatographic methods on different solid support matrices.

The preferred isolated Ixolaris and Ixolaris-related amino acidsequences of the present invention may be synthesized by standardmethods known in the chemical arts.

The isolated amino acid sequences of the present invention may beprepared using solid-phase synthesis, such as that described byMerrifield, 1964 J Amer Chem Soc 85:2149 or other equivalent methodsknown in the chemical arts, such as the method described by Houghten1985 PNAS USA 82:5132 (1985).

Alternatively, the preferred isolated Ixolaris and Ixolaris-relatedamino acid sequences of the present invention may be made by recombinantDNA methods taught herein and well known in the biological arts(Sambrook et al. 1989 Molecular Cloning, A Laboratory Manual, ColdSprings Harbor Press, N.Y.; Ausubel et al. 1989 Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y.). Such methods can be used to construct expression vectorscontaining the cDNA and other nucleotide sequences described in thesection above and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.

Methods

Provided in certain aspects are methods of inhibiting growth of a cellexpressing tissue factor (TF), comprising contacting the cell with aneffective amount of a tissue factor pathway inhibitor (TFPI) compound,such that the growth of the cell is inhibited.

Also provided in other aspects are methods of treating or preventing aTF mediated or associated disease or process in a subject, comprisingadministering to the subject a TFPI compound in an amount effective totreat or prevent the TF mediated or associated disease or process.

Provided in other certain aspects are methods of treating or preventingthe growth or metastasis of tumor cells in a subject, comprisingadministering to the subject a TFPI compound in an amount effective totreat or prevent the growth or metastasis of the tumor cells.

In certain preferred embodiments, the TFPI compound comprises a ticksaliva protein.

Preferably, the TFPI compound comprises at least an active fragment ofan Ixolaris polypeptide.

The present invention contemplates treating or preventing a TF mediatedor associated disease or process in a subject.

In preferred embodiments, the disease is a vascular disease.

Accordingly, the invention features a method of treating or preventing avascular disease in a subject comprising administering to the subject aTFPI compound in an amount effective to treat or prevent the vasculardisease.

Vascular disease includes any disease or disorder that affects thecirculatory system, and can be a disease of the arteries, veins andlymph vessels to blood disorders that affect circulation. Examples ofvascular diseases include, but are not limited to, Peripheral ArteryDisease (PAD), diabetic retinopathy, age-related macular degeneration,aneurysm, renal artery disease, Raynaud's Phenomenon (also calledRaynaud's Disease or Raynaud's Syndrome), Buerger's Disease, PeripheralVenous Disease, Varicose Veins, Venous Blood Clots, Deep veinthrombosis.

In certain preferred embodiments, the disease is cancer.

The cancer to be treated according to the present invention can be asolid tumor or a non-solid tumor (e.g., blood tumors such as leukemias).

The cancer can be metastatic.

Cancer can occur in nearly any tissue of the body including, but notlimited to, adrenocortical carcinoma, anal cancer, bladder cancer, brainstem glioma, brain tumors, breast cancer, cerebellarastrocytoma/malignant glioma, cervical cancer, chronicmyeloproliferative disorders, colon cancer, endometrial cancer,ependymoma, esophageal cancer, Ewing family of tumors, extracranial germcell tumors, extragonadal germ cell tumors, extrahepatic bile ductcancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoidtumors, gestational trophoblastic tumors, hypopharyngeal cancer, isletcell carcinoma (Endocrine Pancreas), islet cell tumors (endocrinepancreas), laryngeal cancer, leukemia, acute lymphoblastic; leukemia,acute myeloid; leukemia, chronic lymphocytic; leukemia, chronicMyelogenous, lip and oral cavity cancer, liver cancer, lung cancer,non-small cell; lung cancer, small cell, lymphoma, Hodgkin's, lymphoma,Non-Hodgkin's, lymphoma, AIDS-Related, lymphoma, lymphoma, primary CNS,melanoma, intraocular (Eye), medulloblastoma, Merkel cell carcinoma,mesothelioma, mycosis fungoides and the Sézary Syndrome, myelodysplasticand myeloproliferative diseases, myelodysplastic Syndromes, myeloidleukemia/other myeloid cancers, nasopharyngeal cancer, neuroblastoma,oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma ofbone, ovarian epithelial cancer, ovarian germ cell tumors, pancreaticcancer, paranasal sinus and nasal cavity cancer, parathyroid cancer,penile cancer, pheochromocytoma, pituitary tumors, prostate cancer,rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma,salivary gland cancer, sarcoma, soft Tissue, sarcoma, Kaposi, skincancer, small Intestine cancer, squamous neck cancer with occultprimary, supratentorial primitive neuroectodermal tumors andpineoblastoma, testicular cancer, thymoma and thymic carcinoma, thyroidcancer, transitional cell cancer of the renal pelvis and ureter,urethral cancer, uterine sarcoma, vaginal cancer, visual pathway andhypothalamic glioma, and vulvar cancer.

Preferably, the cancer comprises a tumor with high expression orproduction of one or more proteins selected from the group consisting oftissue factor (TF), Factor VIIa, Factor Xa, thrombin, vascularendothelial growth factor (VEGF), interleukin-8 (IL-8), one or morematrix metalloproteases, Factor VII, and Factor X, as compared to acontrol cell not derived from the tumor.

In further embodiments, the cancer comprises a tumor wherein the tumorexpresses TF around the necrotic core.

In other further embodiments, the tumor is a highly vascularized tumor.

In preferred embodiments, the tumor comprises a central nervous systemtumor. Astrocytomas and oligodendrogliomas are the most common primarytumors of the adult brain. Both tumors are types of gliomas. Primarybrain tumors arise from cells of the brain itself rather than traveling,or metastasizing, to the brain from another location in the body.Gliomas can be slowly growing (low-grade, grades 1 and 2), or rapidlygrowing (high-grade, grades 3 and 4).

In preferred embodiments, the central nervous system tumor is aglioblastoma.

Glioblastoma multiforme is the most aggressive of the gliomas, acollection of tumors arising from glia or their precursors within thecentral nervous system. Clinically, gliomas are divided into fourgrades; unfortunately, the most aggressive of these, grade 4 orglioblastoma multiforme (GBM), is also the most common in humans. Mostpatients with GBMs die of their disease in less than a year andessentially none has long-term survival.

In other certain preferred embodiments, the tumor comprises a squamouscell tumor, for example a melanoma. Melanoma, the most serious type ofskin cancer, develops in the cells that produce melanin, the pigmentthat gives your skin its color. Melanoma can also form in the eyes and,more rarely, in internal organs, such as your intestines. Skin cancer isthe most common cancer in the United States, and continues to rapidlyincrease. Although other forms of skin cancer, such as basal cell andsquamous cell carcinomas, are on the rise, the greatest increase hasbeen in melanoma. Most melanomas appear without any accompanyingsymptoms. Approximately 70 percent of these cancers arise fromnormal-appearing skin, while the remaining 30 percent arise from anexisting mole. If left untreated, the tumor can spread downward intodeeper skin layers, and to lymph nodes and internal organs.

Methods of the present invention encompass treating or preventing thegrowth of tumor cells. Preferably, in certain embodiments, treating orpreventing the growth of tumor cells comprises at least one selectedfrom the group consisting of decreasing the rate of tumor growth,stopping tumor growth, shrinking the tumor, lessening tumor burden,preventing metastasis, or reducing at least one sign or symptomassociated with the presence of a tumor.

In related embodiments, one or more tumor markers are a sign or symptomassociated with the presence of a tumor.

As described herein, in the methods of the present invention, the TFPIcompound comprises a tick saliva protein. In further preferredembodiments, the TFPI compound comprises at least an active fragment ofan Ixolaris polypeptide.

In certain methods of the invention, administering a tick saliva proteininhibits angiogenesis in the subject.

Angiogenesis is meant to refer to the process of forming new bloodvessels in a subject.

Angiogenesis is involved in many diseases. For example, cardiovasculardiseases such as angioma, angiofibroma, vascular deformity,atherosclerosis, synechia and edemic sclerosis; and opthalmologicaldiseases such as neovascularization after cornea implantation,neovascular glaucoma, diabetic retinopathy, angiogenic corneal disease,macular degeneration, pterygium, retinal degeneration, retrolentalfibroplasias, and granular conjunctivitis are related to angiogenesis.Chronic inflammatory diseases such as arthritis; dermatological diseasessuch as psoriasis, telangiectasis, pyogenic granuloma, seborrheicdermatitis, venous ulcers, acne, rosacea (acne rosacea or erythematosa),warts (verrucas), eczema, hemangiomas, lymphangiogenesis are alsoangiogenesis-dependent.

Tumor angiogenesis is the proliferation of a network of blood vesselsthat penetrates into cancerous growths, supplying nutrients and oxygenand removing waste products. The FDA has approved bevacizumab for usewith other drugs to treat colorectal cancer that has spread to otherparts of the body, some non-small cell lung cancers, and some breastcancers that have spread to other parts of the body. Bevacizumab was thefirst angiogenesis inhibitor proven to delay tumor growth and, moreimportantly, extend the lives of patients. The FDA has also approvedother drugs with antiangiogenic activity as cancer therapies formultiple myeloma, mantle cell lymphoma, gastrointestinal stromal tumors(GIST), and kidney cancer. In certain embodiments of the presentinvention, and anti-angiogenesis agent is administered with a TFPI.

In any of the methods described herein, the method may further compriseidentifying a subject in need of treatment with a TFPI compound.

In any of the methods described herein, the method may further comprisemonitoring a subject for effects of treatment with TFPI compound.

Assessment of the efficacy of a TFPI compound can be determined bymonitoring the subject for amelioration of at least one sign or symptomof cancer.

In certain preferred embodiments, the present invention furthercomprises monitoring the subject for amelioration of at least one signor symptom of cancer.

The TFPI compounds of the present invention can be administered alone,or can be administered in combination concurrently or sequentially withanother agent. For example, the TFPI compounds of the present inventionare used alone, in combination with other compounds of the presentinvention, or in combination with one or more other agents, for example,but not limited to, a cytotoxic agent, an anti-neoplastic agent, animmunosuppressive, and a VEGF antagonist.

For example, the TFPI compounds may be administered with ananti-neoplastic agent, for example, but not necessarily limited toacivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone acetate;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicinhydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguaninemesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin;edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin;enpromate; epipropidine; epirubicin hydrochloride; erbulozole;esorubicin hydrochloride; estramustine; estramustine phosphate sodium;etanidazole; etoposide; etoposide phosphate; etoprine; fadrozolehydrochloride; fazarabine; fenretinide; floxuridine; fludarabinephosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; ilmofosine; interleukin II (includingrecombinant interleukin II, or rIL2), interferon alfa-2a; interferonalfa-2b; interferon alfa-n1; interferon alfa-n3; interferon beta-I a;interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotideacetate; letrozole; leuprolide acetate; liarozole hydrochloride;lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol;maytansine; mechlorethamine, mechlorethamine oxide hydrochloriderethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride, improsulfan, benzodepa, carboquone,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, trimethylolomelamine, chlornaphazine,novembichin, phenesterine, trofosfamide, estermustine, chlorozotocin,gemzar, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol,aclacinomycins, actinomycin F(1), azaserine, bleomycin, carubicin,carzinophilin, chromomycin, daunorubicin, daunomycin,6-diazo-5-oxo-1-norleucine, doxorubicin, olivomycin, plicamycin,porfiromycin, puromycin, tubercidin, zorubicin, denopterin, pteropterin,6-mercaptopurine, ancitabine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, enocitabine, pulmozyme, aceglatone, aldophosphamideglycoside, bestrabucil, defofamide, demecolcine, elformithine,elliptinium acetate, etoglucid, flutamide, hydroxyurea, lentinan,phenamet, podophyllinic acid, 2-ethylhydrazide, razoxane,spirogermanium, tamoxifen, taxotere, tenuazonic acid, triaziquone,2,2′,2″-trichlorotriethylamine, urethan, vinblastine, vincristine,vindesine and related agents. 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cisporphyrin; cladribine; clomifene analogues; clotrimazole; collismycinA; collismycin B; combretastatin A4; combretastatin analogue; conagenin;crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives;curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabineocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine;dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide;dexrazoxane; dexverapamil; diaziquone; didemnin B; didox;diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin;diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; taxel; taxel analogues; taxelderivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin and monoclonal antibodies such as rituximab, trastuzumaband cetuximab.

Pharmaceutical compositions comprising the TFPI compounds of the presentinvention are administered in vivo, ordinarily in a mammal, preferablyin a human. In employing them in vivo, the pharmaceutical compositionscan be administered to a mammal in a variety of ways, including orally,parenterally, intravenously, subcutaneously, intramuscularly,colonically, rectally, nasally or intraperitoneally, employing a varietyof dosage forms. Administration is preferably parenteral, such asintravenous on a daily basis. Alternatively, administration ispreferably oral, such as by tablets, capsules or elixirs taken on adaily basis.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of cell lines, or inanimal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example TFPI compound, preferably a tick saliva protein,preferably an active fragment of Ixolaris polypeptide, which amelioratesthe symptoms or conditions. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD₅₀/dED₅₀.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesare used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

The compositions preferably contain from about 0.01 to 99 weightpercent, more preferably from about 2 to 60 percent, of therapeuticagent together with the adjuvants, carriers and/or excipients.Preferably, the amount of active compound in such therapeutically usefulcompositions is such that a suitable dosage unit will be obtained.Preferred compositions according to the present invention are preparedso that the active fragment of the Ixolaris polypeptide is administeredat a daily dose of about 1 μg/kg to about 1000 μg/kg, about 10 μg/kg toabout 500 μg/kg, about 10 μg/kg to about 750 μg/kg, about 25 μg/kg toabout 1000 μg/kg, about 50 μg/kg to about 1000 μg/kg, about 50 μg/kg toabout 500 μg/kg, about 25 μg/kg to about 500 μg/kg, or about 25 μg/kg toabout 250 μg/kg.

The administration of the TFPI compound can be carried out as frequentlyas required and for a duration that is suitable to provide its effects,e.g. effective treatment for cancer or a vascular disease, or itsunderlying pathological conditions. For example, administration of thetherapeutic agent can be carried out with a single sustained-releasedosage formulation or with multiple daily doses of the therapeuticagent. The amount to be administered will, of course, vary dependingupon the treatment regimen.

In certain preferred embodiments, the active fragment of the Ixolarispolypeptide is administered one time or more, two times or more, threetimes or more, four times or more, five times or more, six times ormore, seven times or more, eight times or more, ten times or more,fifteen times or more, twenty times or more, or twenty five times ormore.

The mammal to be treated in accordance with the present invention can bea rodent, dog, cat, cow, horse, sheep, pig, llama, alpaca, non-humanprimate. Preferably the mammal to be treated is a human.

Gene Therapy

In addition to the administration of therapeutic agents, various genetherapy approaches are contemplated for the methods of the invention,e.g. methods of treating or preventing TF mediated or associateddiseases or processes, e.g. cancer or a vascular disease, as describedabove. In the various gene therapy approaches, a genetic construct isutilized for transformation of cells, preferably patient cells, eitherin vivo or ex vivo. In the latter case the cells are collected from thepatient to be treated, transformed, and then reintroduced into thepatient.

Gene therapy approaches for treating these conditions utilize anexpression vector or plasmid that contains therein a recombinant gene(or genetic construct) encoding a therapeutic protein or nucleic acid.Exemplary therapeutic proteins encoded by the recombinant gene include,without limitation, Ixolaris (Genbank Accession AF286029, which ishereby incorporated by reference in its entirety) and Ixolaris-2(Genbank Accession AY674279, which is hereby incorporated by referencein its entirety), or, for example, a tissue factor pathway inhibitor(TFPI, Genbank Accessions NM 006287 (TFPI var. 1), NM 001032281 (TFPI,var. 2).

The recombinant gene includes, operatively coupled to one another, anupstream promoter operable in mammalian cells and optionally othersuitable regulatory elements (i.e., enhancer or inducer elements), acoding sequence that encodes the therapeutic protein or nucleic acid(described above), and a downstream transcription termination region.Any suitable constitutive promoter or inducible promoter can be used toregulate transcription of the recombinant gene, and one of skill in theart can readily select and utilize such promoters, whether now known orhereafter developed. The promoter can also be specific for expression inpreferred cell types, for example in vascular smooth muscle cells, suchas SM22 (Ribault et al., “Chimeric Smooth Muscle-SpecificEnhancer/Promoters: Valuable Tools for Adenovirus-mediatedCardiovascular Gene Therapy,” Circulation Res. 88(5):468-475 (2001),which is hereby incorporated by reference in its entirety). Tissuespecific promoters can also be made inducible/repressible using, e.g., aTetO response element. Known recombinant techniques can be utilized toprepare the recombinant gene, transfer it into the expression vector,and administer the vector to a patient. Exemplary procedures aredescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Press, N.Y. (1989), which is herebyincorporated by reference in its entirety. One of skill in the art canreadily modify these procedures, as desired, using known variations ofthe procedures described therein.

The recombinant gene can be delivered into targeted cells (to betransformed) as either naked DNA that can be taken up by the cell, or byusing a viral (infective) vector or a non-infective delivery vehicle.

Any suitable viral or infective transformation vector can be used.Exemplary viral vectors include, without limitation, adenovirus,adeno-associated virus, retroviral vectors,

Adenovirus gene delivery vehicles can be readily prepared and utilizedgiven the disclosure provided in Berkner, Biotechniques 6:616-627 (1988)and Rosenfeld et al., Science 252:431-434 (1991), WO 93/07283, WO93/06223, and WO 93/07282, each of which is hereby incorporated byreference in its entirety. Additional types of adenovirus vectors aredescribed in U.S. Pat. No. 6,057,155 to Wickham et al.; U.S. Pat. No.6,033,908 to Bout et al.; U.S. Pat. No. 6,001,557 to Wilson et al.; U.S.Pat. No. 5,994,132 to Chamberlain et al.; U.S. Pat. No. 5,981,225 toKochanek et al.; U.S. Pat. No. 5,885,808 to Spooner et al.; and U.S.Pat. No. 5,871,727 to Curiel, each of which is hereby incorporated byreference in its entirety.

Adeno-associated viral gene delivery vehicles can be constructed andused to deliver into cells a recombinant gene encoding a desired proteinor polypeptide or nucleic acid. The use of adeno-associated viral genedelivery vehicles in vitro is described in Chatterjee et al., Science258:1485-1488 (1992); Walsh et al., Proc. Nat'l Acad. Sci. USA89:7257-7261 (1992); Walsh et al., J. Clin. Invest. 94:1440-1448 (1994);Flotte et al., J. Biol. Chem. 268:3781-3790 (1993); Ponnazhagan et al.,J. Exp. Med. 179:733-738 (1994); Miller et al., Proc. Nat'l Acad. Sci.USA 91:10183-10187 (1994); Einerhand et al., Gene Ther. 2:336-343(1995); Luo et al., Exp. Hematol. 23:1261-1267 (1995); and Zhou et al.,Gene Ther. 3:223-229 (1996), each of which is hereby incorporated byreference in its entirety. In vivo use of these vehicles is described inFlotte et al., Proc. Nat'l Acad. Sci. USA 90:10613-10617 (1993); andKaplitt et al., Nature Genet. 8:148-153 (1994), each of which is herebyincorporated by reference in its entirety.

Retroviral vectors which have been modified to form infectivetransformation systems can also be used to deliver a recombinant geneencoding a desired protein or polypeptide or nucleic acid product into atarget cell. One such type of retroviral vector is disclosed in U.S.Pat. No. 5,849,586 to Kriegler et al., which is hereby incorporated byreference in its entirety.

Alternatively, a number of non-infective delivery vehicles are availablefor delivering the genetic construct in vivo or ex vivo. A colloidaldispersion system can be used to deliver the genetic construct to apatient. Colloidal dispersion systems include macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Thepreferred colloidal system of this invention is a lipid preparationincluding uni-lamellar and multi-lamellar liposomes.

Liposomes are artificial membrane vesicles that are useful as deliveryvehicles in vitro and in vivo. It has been shown that large uni-lamellarvesicles (LUV), which range in size from about 0.2 to about 4.0 .mu.m,can encapsulate a substantial percentage of an aqueous buffer containingDNA molecules (Fraley et al., Trends Biochem. Sci. 6:77 (1981), which ishereby incorporated by reference in its entirety). In addition tomammalian cells, liposomes have been used for delivery ofpolynucleotides in yeast and bacterial cells. For a liposome to be anefficient transfer vehicle, the following characteristics should bepresent: (1) encapsulation of the DNA molecules at high efficiency whilenot compromising their biological activity; (2) substantial binding tohost organism cells; (3) delivery of the aqueous contents of the vesicleto the cell cytoplasm at high efficiency; and (4) accurate and effectiveexpression of genetic information (Mannino et al., Biotechniques 6:682(1988), which is hereby incorporated by reference in its entirety). Inaddition to such LUV structures, multilamellar and small unilamellarlipid preparations which incorporate various cationic lipid amphiphilescan also be mixed with anionic DNA molecules to form liposomes (Felgneret al., Proc. Natl. Acad. Sci. USA 84(21): 7413 (1987), which is herebyincorporated by reference in its entirety).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andtypically the presence of divalent cations. The appropriate compositionand preparation of cationic lipid amphiphile:DNA formulations are knownto those skilled in the art, and a number of references which providethis information are available (e.g., Bennett et al., J. LiposoineResearch 6(3):545 (1996), which is hereby incorporated by reference inits entirety).

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.Examples of cationic amphiphilic lipids useful in formulation ofnucleolipid particles for polynucleotide delivery include the monovalentlipids N-[1-(2,3-dioleoyloxy)propyl]-N,N,N,-trimethyl ammoniummethyl-sulfate, N-[2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammoniumchloride, and DC-cholesterol, the polyvalent lipids LipofectAMINE,dioctadecylamidoglycyl spermine, TRANSFECTAM, and other amphiphilicpolyamines. These agents may be prepared with helper lipids such asdioleoyl phosphatidyl ethanolamine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization. The surface of the targeted delivery system may bemodified in a variety of ways. In the case of a liposomal targeteddelivery system, lipid groups can be incorporated into the lipid bilayerof the liposome in order to maintain the targeting ligand in stableassociation with the liposomal bilayer. Various linking groups can beused for joining the lipid chains to the targeting ligand.

A further alternative for delivery of DNA is the use of a polymericmatrix which can provide either rapid or sustained release of thegenetic construct to the organism. A number of polymeric matrices areknown in the art and can be optimized with no more than routine skill.

The genetic constructs can be used either for direct administration to apatient, in which patient cell transformation occurs in vivo, or for exvivo transformation of previously harvested patient cells that can thenbe reintroduced into the patient to be treated.

Preferred routes for administering a genetic construct for in vivotransformation include (i) administering the genetic construct (aseither an infective vector or as a component within a delivery vehicle)into the right ventricle or a peripheral vein of the patient, and (ii)administering the genetic construct (as a component of a deliveryvehicle) via inhalation. Either of these routes will effectively causethe genetic construct to be delivered into small arterial vessels oflung tissue.

When using ex vivo transformation, it is preferable to harvestprogenitor stem cells from the patient, including progenitor endothelialcells or progenitor vascular smooth muscle cells. After harvesting (andoptionally purifying the population of cells), the harvested progenitorcells are transformed, transformants are selected, and then thetransformed progenitor cells are reintroduced into the lung tissue ofthe patient. Reintroduction of the transformed cells is preferablycarried out by right ventricular administration or peripheralintravenous administration. The ex vivo transformation of endothelialprogenitor cells is described in Zhao et al., “Rescue ofMonocrotaline-Induced Pulmonary Arterial Hypertension Using BoneMarrow-Derived Endothelial-Like Progenitor Cells: Efficacy of CombinedCell and eNOS Gene Therapy in Established Disease,” Circ. Res.96(4):442-450 (2005), which is hereby incorporated by reference in itsentirety).

Antibodies to Ixolaris Proteins

Antibodies that specifically recognize one or more epitopes of Ixolaris,or active fragments thereof, or epitopes of conserved variants ofIxolaris, or peptide fragments of Ixolaris are also encompassed by theinvention. Such antibodies include but are not limited to polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)2 fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above.

The antibodies of the invention may be used, for example, for diagnosticpurposes and for the identification of concentration levels of Ixolarisin various biological fluids. Immunoassays utilizing these antibodiesmay be used as a diagnostic test, such as to detect infection of amammalian host by a tick or to detect Ixolaris from a tick in a tissueof the mammalian host. Also, such immunoassays may be used in thedetection and isolation of Ixolaris from tissue homogenates, clonedcells, and the like.

For the production of antibodies, various host animals may be immunizedby injection with Ixolaris, an Ixolaris peptide (e.g., one correspondingto a functional domain), truncated Ixolaris proteins, polypeptides, orpeptides, functional equivalents of Ixolaris or variants of Ixolaris.Such host animals may include but are not limited to rabbits, mice, andrats, to name but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975 Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al. 1983Immunology Today 4:72; Cole et al. 1983 PNAS USA 80:2026-2030), and theEBV-hybridoma technique (Cole et al. 1985 Monoclonal Antibodies AndCancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may beof any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclass thereof. The hybridoma producing the mAb of this invention maybe cultivated in vitro or in vivo. Production of high titers of mAbs invivo makes this the presently preferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al. 1984 PNAS USA 81:6851-6855; Neuberger etal. 1984 Nature 312:604-608; Takeda et al. 1985 Nature 314:452-454) bysplicing the genes from a mouse antibody molecule of appropriate antigenspecificity together with genes from a human antibody molecule ofappropriate biological activity can be used. A chimeric antibody is amolecule in which different portions are derived from different animalspecies, such as those having a variable region derived from a murinemAb and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird 1988 Science 242:423-426;Huston et al. 1988 PNAS USA 85:5879-5883; and Ward et al. 1989 Nature334:544-546) can be adapted to produce single chain antibodies againstIxolaris gene products. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.1989 Science 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to Ixolaris can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” Ixolaris, using techniques wellknown to those skilled in the art. (See, e.g., Greenspan & Bona 1993FASEB J 7(5):437-444; and Nissinoff 1991 J Immunol 147(8):2429-2438).For example antibodies which bind to a Kunitz domain and competitivelyinhibit the binding of Factor Xa, X, or VIIa to Ixolaris can be used togenerate anti-idiotypes that “mimic” the Kunitz domain and, therefore,bind and neutralize Ixolaris. Such neutralizing anti-idiotypes or Fabfragments of such anti-idiotypes can be used in regimens to neutralizeIxolaris.

Kits

The present invention also features kits for practicing any one of themethods as described herein, e.g. treating or preventing a TF mediatedor associated disease or process in a subject or treating or preventingthe growth or metastasis of tumor cells in a subject, and instructionsfor use.

Preferably, the kits of the present invention comprise a TFPI compound,preferably a tick saliva protein.

In preferred embodiments, the TFPI compound comprises at least an activefragment of an Ixolaris polypeptide.

Kits of the invention may also contain a pharmaceutically acceptablecarrier, a physiologically acceptable carrier, instructions for use, acontain, a vessel for administration, an assay substrate, or anycombination thereof.

EXAMPLES

It should be appreciated that the invention should not be construed tobe limited to the examples that are now described; rather, the inventionshould be construed to include any and all applications provided hereinand all equivalent variations within the skill of the ordinary artisan.

Example 1 Functional TF expressed by U87-MG cells is inhibited byIxolaris

Previous studies employing the U87-MG human glioma cell line havedemonstrated constitutive expression of the clotting initiator proteintissue factor (TF) [20,33]. Accordingly, FIG. 1(A, left) demonstratespositive staining for TF on U87-MG cells, as assessed by flow-cytometricanalysis. In fact, comparison with the human breast cancer cell lineMDA-MB-231 (FIG. 1A, right) showed that U87-MG express moderate levelsof TF. Further enzymatic assays showed that the TF expressed on U87-MGcells is functional. FIG. 1(B) shows that FXa formation was both cell-and FVIIa-dependent, indicating the formation of the FVIIa/TF complex(extrinsic tenase complex).

Ixolaris is a potent inhibitor of the FVIIa/TF complex that blocks FXaformation by forming a quaternary FVIIa/TF/FX/Ixolaris complex in whichthe FVIIa catalytic site is inactivated [24]. Therefore, it was nextdetermined whether Ixolaris inhibits the extrinsic tenase complexassembled on U87-MG cells. As shown in FIG. 1(C), Ixolaris efficientlydecreased FXa formation in this cell-based system.

Example 2 Ixolaris Inhibits PS-Dependent Procoagulant ComplexesAssembled on U87-MG Cells

Previous studies demonstrate that viable tumor cells may expose theanionic phospholipid phosphatidylserine (PS) at the outer leaflet of thecell membrane [31,34]. This ability allows an alternative pathway foractivation of FX through assembly of the intrinsic tenase complex (i.e.FIXa, FVIIIa and PS containing membranes). The presence of PS on thesurface of U87-MG cells was demonstrated by flow cytometric assays usingannexin-V-FITC (FIG. 2A) and specific anti-PS antibodies (data notshown). This indicates that U87-MG cells normally expose PS on theirsurface. The ability of tumor cells to promote FX activation throughassembly of the intrinsic tenase complex was further investigated. FIG.2(B) shows that zymogen activation was both cell- and FVIIIa-dependent.Thus, U87-MG glioma cells support the formation of the intrinsic tenasecomplex.

Assembly of the prothrombinase complex on tumor cells is also supportedby PS exposure [31,35]. Accordingly, FIG. 2(C) shows that U87-MG cellspotentiate prothrombin activation in the presence of FXa and FVa, itsprotein cofactor. On the other hand, no thrombin formation has beenobserved in the absence of cells or in the absence of FVa. These dataare consistent with the assembly of the prothrombinase complex on U87-MGcells. The contribution of tumor cell PS to either FXa or thrombinformation was reinforced by the observation that increasing annexin Vconcentrations progressively decreased zymogen conversion by theirrespective U87-MG-assembled activating complexes (FIG. 2D).

It has been demonstrated that binding of Ixolaris to FX decreases thezymogen recognition by the intrinsic tenase complex, as demonstrated ina purified system [27]. Similarly, increasing Ixolaris concentrationsreduced FXa formation by the U87-MG-assembled intrinsic complex (FIG.2E). Remarkably, effective Ixolaris concentrations in this assay arecorrelated with zymogen concentration and are expected to be much higherthan that required to inhibit the FVIIa/TF complex. Binding of Ixolaristo FXa occurs through a specific heparin binding region in the enzymethat is crucial for prothrombinase complex activity [26]. Therefore,Ixolaris progressively decreased thrombin formation by theU87-MG-assembled prothrombinase complex, as observed in FIG. 2F.

Example 3 Procoagulant Activity of U87-MG Cells is Reversed by Ixolaris

Because U87-MG cells contain the main components to initiate (TF) and topropagate (a PS-containing membrane) blood clotting in a highlyefficient manner, the effect of these cells on human plasma clotting wasfurther tested. As shown in FIG. 3(A), increasing cell concentrationsconsiderably accelerates the coagulation time, demonstrating that thesecells display potent procoagulant activity. However, TF seems to beimportant, if not critical, for this ability, because DEGR-FVIIa, whichacts as a specific TF/FVIIa inhibitor, completely reversed U87procoagulant activity (FIG. 3B, grey bars). The ability of Ixolaris toinhibit the tumor-dependent procoagulant activity was further examined.As expected, Ixolaris efficiently reversed tumor-induced plasmacoagulation (FIG. 3B, black bars).

Example 4 Ixolaris Inhibits In Vivo Primary Tumor Growth in a XenograftModel

It has been demonstrated that coagulation inhibitors targeting theFVIIa/TF complex reduce primary tumor growth and tumor vessel density[36,37]. In this context, the ability of Ixolaris to interfere with invivo U87-MG growth was next examined using a xenograft model in nudemice. As may be seen in FIG. 4, treatment with Ixolaris decreased tumorgrowth progression in a dose-dependent manner (FIG. 4A) with significantreduction in tumor weight in animals treated with either 50 or 250 μgkg⁻¹ (FIG. 4B). In vitro assays for cell proliferation and viabilityshowed no direct toxic effect of Ixolaris on tumor cells (data notshown). In addition, no bleeding has been observed in controls ortumor-bearing animals treated for up to 30 or 20 days, respectively,with both Ixolaris doses (data not shown).

Example 5 Treatment with Ixolaris Decreases Tumor Angiogenesis

Remarkably, the antitumor effect of Ixolaris was accompanied by asignificant decrease in the VEGF mRNA levels within tumors (FIG. 5A).Further immunohistochemistry analysis confirmed that treatment withIxolaris down regulates VEGF expression (FIG. 5B). Thereforeimmunohistochemistry analysis also confirmed that treatment withIxolaris reduces tumor vessel density, as assessed by CD105 staining(FIG. 5C).

Example 6 Inhibition of TF by Ixolaris Reduces Primary Tumor Growth andMetastasis in a Murine Model of Melanoma

Melanoma is a highly metastatic cancer and there is strong evidence thatTF activity contributes to its aggressive pattern. In this context, ithas been suggested that TF inhibitors may attenuate primary tumor growthand metastasis. In this study we evaluated the effect of Ixolaris, anexogenous TF inhibitor, in a murine model of melanoma employing B16F10cells. TF expression on B16F10 cells was evaluated by flow-cytometry. TFactivity on tumor cells was evaluated by measuring factor X activationby FVIIa. The effects of Ixolaris on primary melanoma growth ormetastasis were evaluated by subcutaneous or intravenous injection ofB16F10 cells in C57/BL6 mice, respectively. VEGF expression on primarytumors was evaluated by immunohistochemistry. Flow-cytometric analysesshowed constitutive TF expression by B16F10 cells. In vitro, murine andhuman tumor-derived TF activity was blocked equally well by Ixolaris.Intravenous co-inoculation of B16F10 cells and Ixolaris (250micrograms/kg) dramatically decreased the number of pulmonary tumornodules (47±10 vs 4±1 in the control group). Further primary growthassays were performed and animals were treated daily with Ixolaris (50or 250 micrograms/kg, i.p.) from day 3 to 18 after s.c. inoculation oftumor cells. A significant decrease in tumor weight was observed forboth Ixolaris doses (28% and 58% respectively) as compared tonon-treated animals. In this regard, immunohistochemistry analysesshowed that inhibition of melanoma growth by Ixolaris is accompanied bya significant down regulation of VEGF and angiogenesis in the tumormass.

The data presented herein demonstrate that Ixolaris targetsB16F10-derived TF resulting in decreased metastatic potential andreduced primary tumor growth and angiogenesis.

It is hypothesized that targeting the blood clotting cascade representsa feasible therapeutic approach for treatment of glioblastoma [23]. Theexamples described herein demonstrate for the first time that Ixolaris,a potent exogenous TF inhibitor, blocks the in vivo growth of humanglioblastoma (U87-MG) cells in a xenograft model. This phenomenon wasaccompanied by a significant decrease in VEGF expression as well asdiminished tumor angiogenesis.

The data presented herein show that Ixolaris is highly efficient ininhibiting the U87-MG-assembled extrinsic tenase complex. Therefore, theantitumor effect of Ixolaris is likely to be attributable to thesuppression of tumor-associated FVIIa/TF complex activity. This issupported by other studies showing that: (i) xenograft models employinga specific anti-human TF showed that suppression of tumor—but nothost-derived TF coagulant activity is sufficient to impair primary tumorgrowth [36,38]; (ii) low-TF mice exhibit unaltered growth ofTF-expressing tumor cell lines, as compared with wild-type mice [39].Most remarkable, tumor progression is also impaired by a TF-directedmonoclonal antibody that specifically suppresses PAR-2-mediatedsignaling in tumor cells without affecting FVIIa/TF complex-mediatedcoagulation [40]. It has been clearly demonstrated that Ixolaris blocksthe FVIIa catalytic site [24] and therefore it possibly suppressesFVIIa/TF complex-mediated signaling in addition to its anti-coagulantunction on tumor cells.

The in vitro data presented herein demonstrates that, in addition to theFVIIa/TF complex, Ixolaris inhibits PS-dependent coagulation complexesassembled on U87-MG cells, through interaction with FX or FXa.Therefore, inhibition of thrombin formation by the prothrombinase mightalso contribute to the antitumor activity of Ixolaris. In this regard,it has been demonstrated that argatroban, a specific thrombin inhibitor,reduces in vivo growth of rat glioblastoma although displaying modestsurvival improvement [41]. At this point, it is possible that therapiestargeting multiple coagulation steps, including the FVIIa/TF complex,may offer better results. In the case of Ixolaris, further studiesemploying an orthotopic model (intracerebral administration of tumorcells) may reinforce this hypothesis. This model will be also importantfor evaluating the risk of intracranial bleeding, which is an importantside-effect of the antithrombotic therapy in glioma patients [42].

There is a high incidence of thrombotic events throughout the course ofmalignant glioma [43]. More recently, tumoral intravascular thrombosiswas reported as a distinguishing feature between glioblastoma—the mostaggressive primary brain tumor—and lower grade astrocytomas [44]. Infact, the prothrombotic properties of glioblastoma cells seem tocontribute to the appearance of hypoxic regions within the tumor [21]and, ultimately, to the formation of necrotic foci that arewell-recognized predictors of poor prognosis [45]. This hypothesis issupported by the observations that: (i) exposure of human glioma celllines to hypoxia markedly increases TF expression, resulting in higherprocoagulant activity [20]; and (ii) patients' specimens show increasedTF expression in cells surrounding the necrotic foci (‘pseudopalisading’cells) [20].

Herein it is demonstrated that the human U87-MG glioblastoma cell linealso displays TF and PS on their surface, resulting in high procoagulantactivity in vitro. Notably, TF is determinant for the in vitro coagulantactivity of U87-MG cells. However, it is possible that in the tumormicroenvironment context, PS contributes in vivo to the elevatedoccurrence of intra-tumoral thrombosis in high-grade gliomas.

Vaso-occlusive and prothrombotic mechanisms seem to be intimatelyrelated to tumor hypoxia, necrosis, and accelerated growth inglioblastoma [21]. In fact, intense angiogenesis is a distinguishingpathological hallmark of glioblastomas relative to lower-grade gliomas.Actually, glioblastoma is one of the most highly vascularized malignanttumors and there is strong evidence that VEGF plays a key role in thisprocess [46]. In this regard, our data show that inhibition ofglioblastoma growth by Ixolaris is accompanied by a significantdownregulation of VEGF and angiogenesis in tumor mass. Given the knownantithrombotic properties of Ixolaris [28], it is possible thatsuppression of angiogenesis derives at least in part, from reduction ofintratumoral thrombosis, which could in turn decrease the hypoxicregions within tumor mass and hypoxia driven VEGF production. Inaddition, other tumor models demonstrate decreased angiogenesis uponinhibition of TF, including carcinoma [36], colorectal [37] and breastcancer [38,40], and melanoma [47].

The involvement of TF on tumor angiogenesis might be coupled toactivation of protease-activated receptors (PARs) by coagulation enzymesgenerated in the tumor microenvironment [17,18]. Binding of FVIIa toTF-expressing tumor cells may elicit signal transduction through PAR-2activation followed by upregulation in VEGF expression [48]. In fact, ithas been recently demonstrated that PAR-2 contributes to the angiogenicswitch during mammary tumor development [49]. On the other hand, Yin etal. [50] have demonstrated that PAR-1 mediates angiogenesis, throughVEGF production, in carcinoma and melanoma models. In addition,thrombin-mediated activation of PAR-1 in U87-MG cells increases VEGFtranscription and expression [51]. Taken together, the effect ofIxolaris on tumor growth and angiogenesis may additionally result fromdecreased activation of PAR-1 and/or PAR-2 in U87-MG cells. Thismechanism remains to be determined.

In conclusion, Ixolaris is a potent anticoagulant that does not producemajor bleeding when injected subcutaneously in different animal models[28,52]. Remarkably, Ixolaris is non-immunogenic molecule (unpublishedobservations) and displays long half-life (>24 h, [28]). In addition,Ixolaris is effective at 50-250 μg kg⁻¹, doses that are two to threeorders of magnitude lower than other molecules affecting TF or thecoagulation cascade in the context of experimental therapeutics forcancer [38,41]. Finally, Ixolaris is an angiogenesis inhibitor thateffectively suppresses tumor vessel formation in vivo. Accordingly,Ixolaris may attenuate the procoagulant state of cancer patients on onehand, and prevent angiogenesis on the other, thus interfering with twoimportant components that contribute to tumor growth and metastasis invivo.

Methods

The above-described examples were carried out with, but not limited onlyto, the methods and materials described below.

Materials

Recombinant Ixolaris was produced in High Five insect cells (Invitrogen,San Diego, Calif., USA), purified, and quantified as previouslydescribed [28]. Human thrombin and prothrombin were purified followingpreviously reported procedures [29]. FXa was purchased from Calbiochem(San Diego, Calif., USA). Human FVa, FX, dansyl-Glu-Gly-Arg (DEGR)-FVIIaand placental annexin V were purchased from Haematologic Technologies(Essex Junction, Vt., USA). Human FIXa and FVIIa were purchased fromAmerican Diagnostica (Greenwich, Conn., USA). Human FVIII (Advate) wasfrom Baxter Healthcare Corporation (Westlake Village, Calif., USA).FVIII was activated with human thrombin. Chromogenic substrates for FXa(S-2765, N-a-benzyloxycarbonyl-D-Arg-Gly-Arg-p-nitroanilide) andthrombin (H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilinedihydrochloride, S-2238) were purchased from Diapharma (Westchester,Ohio, USA).

Cell Culture

The human glioblastoma cell line, U87-MG, was grown at 37° C. in ahumidified, 5% CO2 atmosphere in culture flasks, by subconfluentpassages in Dulbeccos Modified Eagle Medium (DMEM-F12) (GibcoBRL;Invitrogen, Carlsbad, Calif., USA) supplemented with 2 g L⁻¹ HEPES, 60mg L⁻¹ penicillin, 100 mg L⁻¹ streptomycin and 1.2 g L⁻¹ sodiumbicarbonate.

Subconfluent cultures were washed twice with PBS, and cells weredetached with Hank's solution containing 10 mM HEPES and 0.2 mM EDTA,spun at 350×g for 7 min, resuspended in supplemented DMEM-F12 andtransferred at a 1:10 ratio to another culture flask. In allexperiments, cells were resuspended in phosphate buffer-saline (PBS).

Flow Cytometric Analysis

Cells grown in culture were resuspended in phosphate buffered salinecontaining 0.1% BSA and incubated for 15 min, at 4° C., with murinemonoclonal antibodies against human TF (4503, American Diagnostica,Stamford, Conn., USA). After washing to remove unbound antibody, cellswere incubated with goat anti-mouse IgG conjugated with FITC (Santa CruzBiotechnology, Santa Cruz, Calif., USA). Cells were washed again andanalyzed using a FACScalibur (Becton-Dickinson, San Jose', Calif., USA).Data were analyzed using the WINMDI 2.8 version software. For surfacephosphatidylserine detection, U87-MG cells were resuspended in 150 mMNaCl, 5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl2 and 10 mM HEPES, pH 7.3(annexin binding buffer), and incubated with 25 μg mL⁻¹ annexin V-FITC(Molecular Probes; Invitrogen) for 15 min at room temperature. U87-MGcells were also labeled with 10 μg mL⁻¹ propidium iodide (PI) forexclusion of those that had lost plasma membrane integrity, thusbecoming PI permeable.

Factor Xa Generation Assays

Activation of FX to FXa by FVIIa was performed as described [30] in 50mM HEPES, 100 mM NaCl, 5 mM CaCl2, 1 mg mL) 1 BSA, pH 7.5 (HEPES-BSAbuffer), as follows. FVIIa (1 nM) was incubated for different timeperiods, at 37° C., with U87-MG cells (5×10⁵ mL⁻¹ in the presence of 100nM FX. After addition of 50 μL of 300 μM S-2765, prepared in 50 mMTris-HCl, 150 mM NaCl, 10 mM EDTA, 1 mg mL⁻¹ PEG 6,000, pH 7.5(Tris-EDTA buffer), absorbance at 405 nm was recorded, at 37° C., for 20min at 6-s intervals using a Thermomax Microplate Reader (MolecularDevices, Menlo Park, Calif., USA) equipped with a microplate mixer andheating system. Velocities (mOD min⁻¹) obtained in the first minutes ofreaction were used to calculate the amount of FXa formed. Controlsperformed in the absence of cells or in the absence of FVIIa showed nosignificant FXa formation. The inhibitory effect of Ixolaris wasevaluated by preincubating FX with varying amounts of the inhibitor(0-10 nM) for 10 min, at 37° C., prior to adding it to FVIIa (1 nM) andU87-MG cells (5·105 mL)1). FXa formed in the absence of Ixolaris wastaken as 100%. Activation of FX to FXa by FIXa was performed inHEPES-BSA buffer, using a previously described discontinuous assay [31].FIXa (0.2 nM, final concentration) was incubated with FVIIIa (4 IU mL⁻¹,final concentration) in the presence of U87-MG cells (5×10⁵ mL⁻¹) for 5min at 37° C. Reaction was initiated by addition of FX (100 nM, finalconcentration) and aliquots of 25 μL were removed every 2 min and placedinto microplate wells containing 25 μL of Tris-EDTA buffer. Afteraddition of 50 μL of 200 μM S-2765 prepared in Tris-EDTA buffer,absorbance at 405 nm was recorded, at 37° C., for 20 min at 6-sintervals as described above. Negative controls were performed in theabsence of cells or in the absence of FVIIIa, showing no significant FXaformation. In some cases, FX was previously incubated with varyingamounts (0-100 nM) of Ixolaris for 10 min, at 37° C., and FXa formed inthe absence of the inhibitor was taken as 100%.

Thrombin Generation Assay

Activation of prothrombin by the prothrombinase complex (FXa/FVa) wasperformed in HEPES-BSA buffer, using a discontinuous assay [31]. FXa (10pM, final concentration) was incubated with FVa (1 nM, finalconcentration) in the presence of U87-MG cells (5·105 mL)1) for 2 min at37° C. Reaction was initiated by the addition of prothrombin (0.5 μM,final concentration) and aliquots of 10 μL were removed every 1 min intomicroplate wells containing 25 μL of Tris-EDTA buffer. After addition of50 μL of 400 μM S-2238 prepared in Tris-EDTA buffer, absorbance at 405nm was recorded, at 37° C., for 20 min at 6-s intervals using aThermomax Microplate Reader. Velocities (mOD min⁻¹) obtained in thefirst minutes of reaction were used to calculate the amount of thrombinformed. Negative controls were carried out in the absence of cells or inthe absence of FVa, showing no significant thrombin formation.Inhibitory effects of Ixolaris upon the prothrombinase complex weretested by preincubating FXa with varying amounts of Ixolaris (0-4 nM)for 10 min, at 37° C., in HEPES-BSA buffer.

Inhibition of Coagulant Complexes by Annexin V

The effect of annexin V on thrombin or FXa formation was tested asfollows: U87-MG cells (5·105 mL)1) were incubated with varying amountsof annexin V (0-5 nM) for 5 min at 37° C. in HEPES-BSA buffer. Cellswere then incubated with either FXa (10 pM)/prothrombin (0.5 lM), orFIXa (0.2 nM)/FX (100 nM) followed by the addition of FVa (1 nM) orFVIIIa (4 IU mL⁻¹), respectively. Aliquots of 25 μL were removed after 2min, and delivered to microplate wells containing 25 μL of Tris-EDTAbuffer. The amount of FXa or thrombin formed was evaluated as describedabove, using S-2238 or S-2765, respectively.

Procoagulant Activity Measured by Recalcification Time

The ability of U87-MG glioblastoma cells to potentiate plasmacoagulation was assessed by measuring the recalcification time on anAmelung KC4A coagulometer (Labcon, Heppenheim, Germany) using plastictubes. Human blood samples were collected from healthy donors in 3.8%trisodium citrate (9:1, v/v), and platelet-poor plasma was obtained byfurther centrifugation at 2000×g for 10 min. Plasma was incubated with50 μL of U87-MG glioblastoma cells at various concentrations (suspensionin TBS buffer) for 1 min at 37° C. Plasma clotting was initiated by theaddition of 100 μL of 12.5 mM CaCl₂ and the time for clot formation wasthen recorded. In some cases, cells were previously incubated with theinactivated form of FVIIa, DEGR-FVIIa, for 10 min at room temperature.The in vitro effect of Ixolaris on U87-MG-induced coagulation wasevaluated using the following procedure: plasma (50 μL) was incubatedwith Ixolaris (10 μL) for 10 min at 37° C., followed by the addition of100 lL of 12.5 mM CaCl2.

Tumor Growth Assay

U87-MG cells (2×10⁶) were subcutaneously inoculated into the flank of6-week-old, male Balb/C nude mice (Chemistry Institute Animal Room, SaoPaulo University, Sao Paulo, Brazil). Treatment with Ixolaris (dilutedin PBS, 100 μL final volume) was initiated 3 days after tumor cellinoculation and continued daily for 17 days. Control animals receivedPBS instead of the inhibitor. Treatment was performed by subcutaneousadministration into the flank, preferentially at distant sites fromtumor inoculation. Tumor growth was evaluated for 20 days with calipers,and the volume was calculated using the equation:(length)×(width)·(π/6). Preliminary analysis showed macroscopic necroticareas in some control animals after 20 days of cell inoculation. Tumorweight was determined at the time of sacrifice. Analysis of variance(ANOVA) was used to establish differences between groups, andsignificance levels were determined by nonparametric Mann-Whitney test.Animal experiments were performed under approved protocols of theinstitutional animal use and care committee.

RNA Isolation and Reverse Transcriptase Real-Time PCR

Tumor RNA was isolated using the Trizol reagent (Invitrogen). After cDNAsynthesis using Superscript III reverse transcriptase (Invitrogen), mRNAlevels were determined by quantitative polymerase chain reaction (PCR)on a Gene-Amp 7300 Sequence Detection System (Applied Biosystems, FosterCity, Calif., USA) using SYBR Green Master mix and sequence-specificprimers designed using Primer Express 3 (Applied Biosystems). Primersused were: VEGF (F: 5′-AGTGGTGAAGTTCATGGATGT-3′, R:5′-GCACACAGGATGGCTTGAAGA-3′) and GAPDH (F: 5′-CCCACTCCTCCACCTTTGA-3′, R:5′-CTGTTGCTGTAGCCAAA TTCGT-3′).

Immunohistochemistry

Tissue staining was performed on paraffin-embedded sections (4-lmthick), which were incubated overnight, following heat antigenretrieval, with primary antibodies: monoclonal antimouse endoglin(CD105) antibody (MAB-1320, R&D Systems, Minneapolis, Minn., USA) at1:20 dilution, or monoclonal antibody against VEGF (SC-7269, Santa CruzBiotechnology) at 1:100 dilution. In order to reduce non-specificantibody binding, sections were incubated with PBS containing 10%non-immune goat serum, 5% BSA and 10% fetal bovine serum for 30 minprior to incubation with primary antibodies. Sections were furtherrevealed using the LSAB2 Kit, HRP (Dako-Cytomation, Carpinteria, Calif.,USA) with diaminobenzidine (3,3′-diaminobenzidine tablets; SigmaChemical Co, St Louis, Mo., USA) as the chromogen and counterstainedwith hematoxylin. Negative control slides consisted of sectionsincubated with antibody vehicle or non-immune rat or mouse serum. Tenfields of immunostained section (CD105 and VEGF) were chosen at randomand captured from each specimen. Quantification was assessed on capturedhigh quality images (2048. 1536 pixels buffer) using IMAGE PRO PLUS4.5.1 (Media Cybernetics, Silver Spring, Md., USA). Data were stored inAdobe Photoshop, version 3.0 (Adobe Systems Inc., San Jose, Calif.,USA), to enable uneven illumination and background color to becorrected. The number of transversal sections of CD105 was counted, andthese numbers per square millimeter of the tumor were calculated, aspreviously described [32]. A semi-quantitative evaluation ofimmunohistochemical staining for VEGF was performed as described [32].Statistical analyses comparing control and treatment groups used one-wayANOVA. Values of P<0.05 were considered to be statistically significant.

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INCORPORATION BY REFERENCE

The contents of all references, patents, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of inhibiting growth of a cell expressing tissue factor(TF), comprising contacting the cell with an effective amount of atissue factor pathway inhibitor (TFPI) compound, such that the growth ofthe cell is inhibited.
 2. A method of treating or preventing a TFmediated or associated disease or process in a subject, comprisingadministering to the subject a TFPI compound in an amount effective totreat or prevent the TF mediated or associated disease or process.
 3. Amethod of treating or preventing the growth or metastasis of tumor cellsin a subject, comprising administering to the subject a TFPI compound inan amount effective to treat or prevent the growth or metastasis of thetumor cells.
 4. The method of claim 1, wherein the cells are selectedfrom the group consisting of: tumor cells, endothelial cells, vascularsmooth muscle cells, inflammatory cells, or a combination thereof. 5.The method of any one of claims 1-3, wherein the TFPI compound comprisesa tick saliva protein.
 6. The method of claim 4, wherein the TFPIcompound comprises at least an active fragment of an Ixolarispolypeptide.
 7. The method of claim 2, wherein the disease is selectedfrom the group consisting of: cancer and a vascular disease.
 8. Themethod of claim 3, wherein the tumor comprises a solid tumor.
 9. Themethod of claim 3, wherein the tumor comprises a central nervous systemtumor or a squamous cell tumor.
 10. The method of claim 8, wherein thetumor is a glioblastoma.
 11. The method of claim 9, wherein the tumor isa melanoma.
 12. The method of any one of claims 8-11, wherein the tumoris a metastatic tumor.
 13. The method of claim 3, wherein treating orpreventing the growth of tumor cells comprises at least one selectedfrom the group consisting of: decreasing the rate of tumor growth,stopping tumor growth, shrinking the tumor, lessening tumor burden,preventing metastasis, or reducing at least one sign or symptomassociated with the presence of a tumor.
 14. The method of claim 13,wherein one or more tumor markers are a sign or symptom associated withthe presence of a tumor.
 15. A method of treating or preventing avascular disease in a subject comprising administering to the subject aTFPI compound in an amount effective to treat or prevent the vasculardisease.
 16. The method of claim 15, wherein the TFPI compound comprisesa tick saliva protein.
 17. The method of claim 16, wherein the TFPIcompound comprises at least an active fragment of an Ixolarispolypeptide.
 18. The method of claim 15, wherein the vascular disease isselected from the group consisting of: diabetic retinopathy, age-relatedmacular degeneration, and pulmonary hypertension.
 19. The method ofclaim 15, wherein administering a tick saliva protein inhibitsangiogenesis in the subject.
 20. The method of any one of claim 1-3 or15, wherein the TFPI compound is administered in combinationconcurrently or sequentially with another agent.
 21. The method of claim20, wherein the agent is selected from the group consisting of: acytotoxic agent, an anti-neoplastic agent, an immunosuppressive, and aVEGF antagonist.
 22. The method of any one of claim 1-3 or 15, furthercomprising identifying a subject in need of treatment with a TFPIcompound.
 23. The method of any one of claim 1-3 or 15, furthercomprising monitoring a subject for effects of treatment with TFPIcompound.
 24. A method of treating or preventing cancer in a subjectcomprising administering to the subject a TFPI compound in an amounteffective to treat or prevent cancer.
 25. The method of claim 24,wherein the TFPI compound comprises a tick saliva protein.
 26. Themethod of claim 25, wherein the TFPI compound comprises at least anactive fragment of an Ixolaris polypeptide.
 27. The method of claim 23or 24, further comprising monitoring the subject for amelioration of atleast one sign or symptom of cancer.
 28. The method of any one of claim7 or 24, wherein the cancer comprises a tumor with high expression orproduction of one or more proteins selected from the group consistingof: tissue factor (TF), Factor VIIa, Factor Xa, thrombin, vascularendothelial growth factor (VEGF), interleukin-8 (IL-8), one or morematrix metalloproteases, Factor VII, and Factor X, as compared to acontrol cell not derived from the tumor.
 29. The method of any of claim7 or 24, wherein the cancer comprises a tumor wherein the tumorexpresses TF around the necrotic core.
 30. The method of any of claims 1to 24, wherein the tumor is a highly vascularized tumor.
 31. The methodof any of claims 1 to 24, wherein the tumor comprises a glioblastoma.32. The method of any of claims 1 to 24, wherein an active fragment ofan Ixolaris polypeptide comprises at least 40 contiguous amino acids ormore, 50 contiguous amino acids or more, 60 contiguous amino acids ormore, 70 contiguous amino acids or more, 80 contiguous amino acids ormore, 90 contiguous amino acids or more, 100 contiguous amino acids ormore, 110 contiguous amino acids or more, 120 contiguous amino acids ormore, 130 contiguous amino acids or more, 140 contiguous amino acids ormore, 150 contiguous amino acids or more, 160 contiguous amino acids ormore, or the full length sequence of the amino acid sequencecorresponding to SEQ ID NO:
 2. 33. The method of any of claims 1 to 24,wherein an active Ixolaris polypeptide comprises at least 80% overallidentity or more, 85% overall identity or more, 90% overall identity ormore, 95% overall identity or more to a fragment of at least 50contiguous amino acids of SEQ ID NO:
 2. 34. The method of claim 27,wherein amelioration of at least one sign or symptom of cancer comprisesat least one of a reduction in tumor volume or a reduction of expressionor production of at least one of tissue factor (TF), Factor VIIa, FactorXa, thrombin, vascular endothelial growth factor (VEGF), IL-8, one ormore matrix metalloproteases, Factor VII, or Factor X as compared toprior to treatment with an Ixolaris polypeptide.
 35. The method of claim34, wherein a reduction in tumor volume comprises a reduction of 20% ormore, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more,80% or more, or 90% or more.
 36. The method of any of claims 1 to 24,wherein the active fragment of an Ixolaris polypeptide comprisesadministration of a nucleic acid encoding the active fragment of anIxolaris polypeptide operably linked to control sequences for expressionof the polypeptide.
 37. The method of any of claims 1 to 24, wherein themethod further comprises administration of a an agent for treatment ofexcess angiogenesis.
 38. The method of any of claims 1 to 24, furthercomprising obtaining an Ixolaris polypeptide.
 39. The method of any ofclaims 1 to 24, wherein the active fragment of the Ixolaris polypeptideis administered at a daily dose of about 1 μg/kg to about 1000 μg/kg,about 10 μg/kg to about 500 μg/kg, about 10 μg/kg to about 750 μg/kg,about 25 μg/kg to about 1000 μg/kg, about 50 μg/kg to about 1000 μg/kg,about 50 μg/kg to about 500 μg/kg, about 25 μg/kg to about 500 μg/kg, orabout 25 μg/kg to about 250 μg/kg.
 40. The method of any of claims 1 to24, wherein the active fragment of the Ixolaris polypeptide isadministered one time or more, two times or more, three times or more,four times or more, five times or more, six times or more, seven timesor more, eight times or more, ten times or more, fifteen times or more,twenty times or more, or twenty five times or more.
 41. The method ofany of claims 1 to 24, wherein the Ixolaris polypeptide is an isolatedpolypeptide.
 42. A pharmaceutical composition for practicing any of themethods of claims 1 to
 24. 43. A pharmaceutical composition comprisingan isolated Ixolaris polypeptide in a pharmaceutical excipient.
 46. Anantibody that recognizes one or more epitope of a TFPI compound.
 47. Theantibody of claim 46, wherein the TFPI compound comprises a tick salivaprotein.
 48. The antibody of claim 47, wherein the TFPI compoundcomprises at least an active fragment of an Ixolaris polypeptide.
 49. Akit for practicing any of the methods of claims 1 to 24, andinstructions for use.
 50. The kit of claim 49, wherein the kit comprisesa TFPI compound.
 51. The kit of claim 50, wherein the TFPI compoundcomprises a tick saliva protein.
 52. The kit of claim 51, wherein theTFPI compound comprises at least an active fragment of an Ixolarispolypeptide.